Serum half-life extension using igbd

ABSTRACT

The present invention relates to complexes comprising (i) an immunoglobulin (Ig) binding moiety and (ii) a pharmaceutically active moiety, wherein the Ig binding moiety specifically binds to the constant domain 1 of the heavy chain (C H 1) of an Ig molecule and their use for therapy and prophylaxis.

The present invention relates to complexes comprising (i) animmunoglobulin (Ig) binding moiety and (ii) a pharmaceutically activemoiety, wherein the Ig binding moiety specifically binds to the constantdomain 1 of the heavy chain (C_(H)1) of an Ig molecule and their use fortherapy and prophylaxis.

BACKGROUND

Most of the therapeutic applications of pharmaceuticals benefit frommaintaining a therapeutic effective concentration over a prolongedperiod of time, often requiring a frequent administration or infusions,or a loco-regional application or subcutaneously of the drug utilizing aslow adsorption into the blood stream in order to maintain an effectiveconcentration over a prolonged period of time. When a drug isadministered by rapid intravenous injection into the vascular system,its removal from the blood almost always occurs in a biphasic fashion(see Greenblatt (1985) Ann. Rev. Med. 36:421-427). This can bemathematically described by a two-compartment model, which resolves thebody into a central compartment and a peripheral compartment (seeDhillon and Gill: Basic Pharmacokinetics). These compartments have nodistinct physiological or anatomical delimitation, however, the centralcompartment is considered to comprise tissues that are highly perfused(e.g. heart, lungs, kidneys, liver and brain) whilst the peripheralcompartment comprises less well-perfused tissues (e.g. muscle, fat andskin). A two-compartment model assumes that upon drug administrationinto the central compartment, e.g. into the blood stream, the drugdistributes between the central compartment and the peripheralcompartment. However, the drug does not achieve instantaneousdistribution, i.e. equilibration, between the two compartments. The drugconcentration-time profile shows a curve, with the log drugconcentration-time plot showing a biphasic response which can be used todistinguish whether a drug shows a one- or two-compartment model (seeDhillon and Gill: Basic Pharmacokinetics). Immediately after the dose isgiven, there is a phase of rapid drug disappearance from the blood,usually lasting from a few minutes to an hour or two, which may lead toa very substantial decrement in drug concentrations in blood. Thisinitial phase (described by the initial or distribution plasmahalf-life; t_(1/2)α) of rapid drug disappearance is determined mainly byreversible distribution of drug out of the “central” compartment, ofwhich the vascular system is a component, into storage sites inperipheral tissues; very little of this initial rapid decline isdetermined by elimination or clearance. After distribution is complete,the blood concentration curve enters a less rapid phase of drugdisappearance, termed the elimination phase (described by the terminalor elimination plasma half-life; t_(1/2)β), during which drugdisappearance is determined mainly by irreversible clearance. Thepattern of drug decline during this elimination phase is used tocalculate the elimination plasma half-life which is generally determinedonly after drug distribution equilibrium has been attained (seeGreenblatt (1985) Ann. Rev. Med. 36:421-427). Both, the initial plasmahalf-life and the terminal plasma half-life of a substance, e.g. apharmaceutical, can be influenced in order to extend the bioavailabilityof such substance in the body by preventing its rapid clearance from theblood.

Small molecule pharmaceuticals, in particular most small proteintherapeutics, including many of the alternative recombinant antibodyformats (Kontermann (2010) Curr. Opin. Mol. ther. 12:176-183) but alsothe emerging class of alternative scaffold proteins (Nuttall & Walsh(2008) Curr. Opin. Pharmacol. 8:609-615; Gebauer & Skerra (2009) Curr.Opin. Chem. Biol. 13:245-255), suffer from a short serum half-lifemainly due to their rapid clearance from circulation (Batra et al.(2002) Curr. Opin. Biotechnol. 13:603-608). These limitations of smallsize drugs has led to the development and implementation of half-lifeextension strategies to prolong circulation of these recombinantantibodies in the blood and thus to improve administration andpharmacokinetic as well as pharmacodynamic properties.

Extension of the half-life can help to reduce the number of applicationsand to lower doses, thus are beneficial for therapeutic but alsoeconomic reasons. Strategies to extend the plasma half-life ofpharmaceuticals and therapeutic proteins have, therefore, attractedincreasing interest (Pisal et al., (2010) J. Pharmaceut. Sci.99:2557-2575; Kontermann (2009) BioDrugs 23:93-109; Kontermann (2011)Curr. Opin. Biotechnol. in press).

Several mechanisms are involved in clearance of drugs from circulationincluding peripheral blood-mediated elimination by proteolysis, renaland hepatic elimination, and elimination by receptor-mediatedendocytosis (Tang et al. (2004) J. Pharmaceut. Sci. 93:2184-2204).Molecules possessing a small size, i.e. a low molecular mass with athreshold in the range of 40-50 kDa, are rapidly cleared by renalfiltration and degradation. Responsible for renal clearance is theglomerular filtration barrier (GBM) formed by the fenestratedendothelium, the glomerular basement membrane and the slit diaphragmlocated between the podocyte foot processes (Tryggvason & Wartiovaara(2005) Physiology 20:96-101). While the fenestrae between the glomerularendothelial cells are rather large (50-100 nm) allowing free diffusionof molecules, the slit diaphragm represents the ultimate macromolecularbarrier, forming an isoporous, zipper-like filter structure withnumerous small, 4-5 nm diameter pores and a lower number of 8-10 nmdiameter pores (Haraldsson & Sorensson (2004) New Physiol. Sci. 19:7-10;Wartiovaara et al. (2004) J. Clin. Invest. 114:1475-1483). Moleculeswith a hydrodynamic radius smaller than approximately 4-5 nm aretherefore rapidly cleared from the blood. In addition, the charge of aprotein contributes to renal filtration. Proteoglycans of theendothelial cells and the GBM form an anionic barrier, which partiallyprevents the traversal of negatively charged plasma macromolecules(Tryggvason & Wartiovaara (2005) Physiology 20:96-101). Consequently,the size of a protein therapeutic, i.e. its hydrodynamic radius, butalso its physicochemical properties represent starting points in orderto improve half-life. Furthermore, some plasma proteins such as serumalbumin and IgG molecules possess an extraordinary long half-life in therange of 2-4 weeks in humans, which clearly discriminates thesemolecules from all the other plasma proteins (Kontermann (2009) BioDrugs23:93-109). Responsible is a recycling through the neonatal Fc receptor(FcRn, Brambell receptor) (Roopenian & Akilesh (2007) Nat. Rev. Immunol.7:715-725). Albumin and IgGs taken up by cells, e.g. endothelial cells,through macropinocytosis bind to the FcRn in a pH-dependent manner inthe acidic environment of the early endosome. This binding divergesalbumin and IgG from degradation in the lysosomal compartment andredirects them to the plasma membrane, where they are released back intothe blood plasma due to the neutral pH. This offers additionalopportunities to extend or modulate the half-life of proteins, e.g.through fusion to albumin or the Fc-region of IgG (Kontermann (2009)BioDrugs 23, 93-109). Finally, protein drugs that bind to a cellularsurface receptor will be internalized by receptor-mediated endocytosisand subjected to lysosomal degradation if the protein drug stays boundto the receptor (Tang et al. (2004) J. Pharmaceut. Sci. 93:2184-2204;Lao & Kamei (2008) Biotechnol. Prog. 24:2-7). Hence, engineering of theinteraction of the therapeutic protein with its receptor(s) at acidic pHcan therefore also prolong half-life of the protein by allowingrecycling of the unbound molecules into the blood stream as shown forengineered G-CSF and an anti-IL6 receptor antibody (Sarkar et al. (2002)Nat. Biotechnol. 20:908-913; Igawa et al. (2010) Nat. Biotechnol.28:1203-1208).

Several half-life extension strategies have been developed in recentyears (Kontermann (2009) BioDrugs 23:93-109; Kontermann (2011) Curr.Opin. Biotechnol. in press), including strategies such as PEGylation andhyperglycosylation with the aim to increase the hydrodynamic volume ofthe protein to reduce renal clearance, as well as strategies utilizingrecycling processes executed by the neonatal Fc receptor (FcRn), whichis responsible for the extraordinary long half-lives of serum IgGs andof serum albumin (Kim et al. (2006) Clin. Immunol. 122:146-155). Forexample, albumin has been employed for half-life extension through thegeneration of albumin fusion proteins. Several albumin fusion proteins,e.g. albinterferon alfa-2b and a coagulation factor IX-HSA fusionprotein, have already entered clinical trials (Nelson et al. (2010)Gastroenterology 139:1267-1276; Metzner et al. (2009) Thromb. Haemost.102:634-644). In addition, various molecules exhibiting albumin-bindingactivity have been used for half-life extension. For this approach, thealbumin-binding moiety is coupled or fused to the therapeutic proteinleading to reversible binding to serum albumin after administration.Such albumin-binding molecules include fatty acids, organic molecules,peptides, single-chain Fv, domain antibodies, nanobodies but alsodomains from naturally occurring proteins capable of binding albumin(for review see: Kontermann (2009) BioDrugs 23:93-109). For example, analbumin-binding domain (ABD) from streptococcal protein G was used toprolong the plasma half-life of recombinant antibodies and Affibodymolecules (Stork et al. (2007) Protein Eng. Des. Sel. 20:569-576;Andersen et al. (2010) J. Biol. Chem. 286:5234-5241). Fusion of the ABDresulted in similar half-lives as seen for an albumin fusion protein andan improved tumor accumulation as shown for a bispecific single-chaindiabody (Stork et al. (2007) Protein Eng. Des. Sel. 20:569-576; Stork etal. (2009) J. Biol. Chem. 284:25612-25619). These studies, however, alsorevealed that albumin and ABD fusion proteins do not reach the longhalf-life of IgG molecules. Attempts to further prolong half-life byapplying an ABD with increased affinity for albumin resulted only in amarginal improvement (Hopp et al., 2010, Protein Eng. Des. Sel.23:827-834). Non-covalent interaction with serum IgG also represents afeasible alternative to binding to serum albumin. This approach wasalready tested with a bispecific diabody with affinity for mouse Fcγ1,which prolonged the terminal plasma half-life of the diabody from 1.7 hto 10 h in mice (Holliger et al. (1997) Nat. Biotechnol. 15:632-636).

However, many disadvantages are associated with above strategies ofextending the plasma half-life of pharmaceuticals. The usage of PEG,polysialic acid and HES requires their chemical conjugation to thepharmaceutical, which consequently complicates the production andanalysis of the final product. PEG is not biologically degradable andmay accumulate in the body of a patient which may lead to furthercomplications. Moreover, it has been shown that these modification wereonly able to prolong the serum half-life of pharmaceuticals to a limitedextend. Similarly, also the serum half-life extension via theconjugation, fusion or binding of the pharmaceutical to serum albumin orvia Fc-fusion proteins remains significantly below the serum half-lifeof IgG. There is thus, a clear need for the development of newstrategies allowing for the extension of the serum half-life ofpharmaceuticals, especially of therapeutic proteins, which overcomethese disadvantages.

Present inventors surprisingly found that the fusion of pharmaceuticalsto an immunoglobulin-binding domain (IgBD) solves this problem. IgBDsare known from various bacterial proteins, e.g. staphylococcal protein A(SpA), streptococcal protein G (SpG) and protein L of Peptostreptococcus(PpL) (Tashiro & Montelione (1995) Curr. Biol. 5:471-481; Sidorin &Solov′eva (2011) Biochemistry (Mosc.) 76:363-378). These IgBDs have alength of 50 to 60 amino acid residues and form either a 3-α-helixbundle or a compact structure composed of a 4-stranded β-sheet and oneα-helix (Tashiro & Montelione (1995) Curr. Biol. 5:471-481). IgBDs are,thus, particularly stable which benefits the production and storageproperties of fusion proteins comprising them.

IgBDs show a high affinity to serum immunoglobulins, with most of thembinding to the same location on the Fc domain of an immunoglobulin asthe neonatal Fc receptor, e.g. in IgG the primary binding site islocated at the C_(H)2-C_(H)3 interface of one heavy chain (Deisenhofer(1981) Biochemistry 20:2361-2370). They are thus, competing with theFcRn binding and may negatively influence the recycling of theimmunoglobulin molecule via the FcRn. For these reasons, IgBDs have sofar not been considered for the extension of the serum half-life ofpharmaceuticals. However, some bacterial IgBDs are also capable ofbinding to different regions of the Fab fragment (Tashiro & Montelione(1995) Curr. Biol. 5:471-481). Present inventors were able to show thatthe fusion of pharmaceuticals to an IgBD significantly prolongs theserum half-life of such pharmaceutical, probably due to the fact thatthese IgBDs do not compete with the Fc receptor binding.

The fusion or conjugation of a pharmaceutical to such IgBD thus,represents an advantageous possibility of extending their serumhalf-life as the binding of such fusion protein to an immunoglobulinmolecule has a twofold effect; firstly, the clearance by renalfiltration and degradation is limited or prevented, and secondly, therecycling of the fusion protein via the FcRn is allowed for.

The complexes of the present invention provide inter alia the followingadvantageous properties increase of the solubility of the respectivepharmaceutically active moiety in vivo, increase of the in vitrostability of the respective pharmaceutically active moiety, whichresults in an extended shelf-life of such fusion protein. In caseswhererin the pharmaceutical active moiety is a protein or peptide, afurther advantage of fusing such moiety to an IgBD of the presentinvention is the increased expression of such fusion proteins, e.g. inmammalian expression systems. In addition, the complexing of thepharmaceutical moiety to an immunoglobulin binding moiety, in particularif the pharmaceutical moiety is a protein or peptide allows an easierand/or faster purification of such pharmaceutical.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a complex comprising(i) an immunoglobulin (Ig) binding moiety and (ii) a pharmaceuticallyactive moiety, wherein the Ig binding moiety specifically binds to theconstant domain 1 of the heavy chain (C_(H)1) of an Ig molecule.

In a second aspect, the present invention provides a nucleic acidmolecule comprising a sequence encoding the complex of the first aspect.

In a third aspect, the present invention provides a vector comprisingthe nucleic acid of the second aspect.

In a fourth aspect, the present invention provides an isolated cellcontaining the complex of the first aspect and/or the nucleic acidmolecule of the second aspect and/or the vector of the third aspect

In a fifth aspect, the present invention provides a pharmaceuticalcomposition comprising the complex of the first aspect, the nucleic acidof the second aspect, the vector of the third aspect and/or the cell ofthe fourth aspect and a pharmaceutical acceptable carrier and/orexcipient.

In a sixth aspect, the present invention provides the complex of thefirst aspects, the nucleic acid of the second aspect, the vector ofthird aspect, the cell of the fourth aspect, the pharmaceuticalcomposition of the fifth aspect for use in extending the serumhalf-life.

In a seventh aspect, the present invention provides the complex of thefirst aspects, the nucleic acid of the second aspect, the vector ofthird aspect, the cell of the fourth aspect, the pharmaceuticalcomposition of the fifth aspect for use as a medicament.

FIGURES

FIG. 1: Summary of IgBD bound to human IgG1. IgBDs from protein A(SpA_(B), SpA_(D)), protein G (SpG_(C2), SpG_(C3)) and protein L(PpL_(C4*)) in complex with IgG were visualized on a human IgG1 model(Clark (1997) Chem. Immunol. 65:88-110). In addition, the extracellularregion of human FcRn bound to the Fc region was included (Burmeister etal. (1994) Nature 372:379-383). PDB entries are indicated for each IgBDand the FcRn. The structures were visualized with the PyMOL MolecularGraphics System (Version 1.3, Schrödinger, LLC). b) Schematicillustration of binding of SpG_(C3), possessing one binding site for CH1and one binding site for the Fc part, to IgG. c) Schematic illustrationof binding of SpG_(C3-Fab), possessing one binding site for CH1 and amutated inactive Fc binding site, to the CH1 domain of IgG only.

FIG. 2: Construction of scDb-IgBDs and scFv-IgBDs. a) Composition of thescDb-IgBD and scFv-IgBD fusion protein. IgBDs are fused to theC-terminus of a bispecific scDb or a scFv. b) SDS-PAGE analysis ofpurified scDb-CEACD3 (1), scDb-SpA_(B) (2), scDb-SpA_(D) (3),scDb-SpA_(EZ4) (4), scDb-SpG_(C3) (5), and scDb-PpL_(C4*) (6) underreducing conditions. c) SDS-PAGE analysis of purified anti-CEA scFv (1),scFv-SpA_(B) (2), scFv-SpA_(D) (3), scFv-SpA_(EZ4) (4), scFv-SpG_(C3)(5), and scFv-PpL_(C4*) (6) under reducing conditions. Two micrograms ofthe proteins were analyzed per lane and the gel was stained withCoomassie brilliant blue G-250 (M, molecular weight standards). d-g)Purified scDb, scFv as well as the scDb-SpG_(C3) and scFv-SpG_(C3)fusion proteins were analyzed by SEC.

FIG. 3. Binding of scDb-IgBD and scFv-IgBD fusion proteins to CEA inELISA. Increasing concentrations of the scDb-IgBD (a) or scFv-IgBD (b)fusion proteins were analyzed for binding to immobilized CEA.

FIG. 4: Binding of scDb-IgBD to IgG, Fab and Fc analyzed by ELISA. scDb,scDb-SpA_(B), scDb-SpA_(D), scDb-SpA_(E4), scDb-SpG_(C3), andscDb-PpL_(C4)* were analyzed for binding to immobilized mouse (a) andhuman (b) serum IgG as well as Fab and Fc fragments thereof.Furthermore, human IgM and human IgA were analyzed for binding of thesefusion proteins.

FIG. 5. Binding of scDb-IgBD to IgG, Fab and Fc analyzed by quartzcrystal microbalance measurements. Human and mouse IgG as well as Faband Fc fragments thereof were immobilized on a QCM chip and binding ofthe scDb-IgBD fusion proteins was determined at 1.5 μM (Fab fragments),500 nM (IgGs), 40 nM (human IgG) or x nM (IgG-Fc fragments), and 1.28 nM(mouse IgG-Fc fragments).

FIG. 6: Affinities. Affinities of the scDb-IgBD fusion proteins forhuman and mouse IgG and Fab and Fc fragments thereof determined atneutral pH (7.4) and acidic pH (6.0) by quartz crystal microbalancemeasurements using an Attana A100 and sensorchips with chemicallyconjugated immunoglobulins.

FIG. 7. Plasma half-life of scDb-IgBD and scFv-IgBD fusion proteins incomparison to unmodified proteins (scDb, scFv) and IgG. ScDb-IgBD (a)and scFv-IgBD (b) fusion proteins were i.v. injected into CD1 mice (25μg/animal) and serum concentrations of the antibody molecules weredetermined at different time points by ELISA. Data were normalizedconsidering maximal concentration at the first time point (3 min).

FIG. 8: Biochemical and pharmacokinetic properties of scDb-IgBD andscFv-IgBD fusion proteins in the mouse. ScDb-IgBD and scFv-IgBD fusionproteins were i.v. injected into CD1 mice (25 μg/animal) and serumconcentrations of the antibody molecules were determined at differenttime points by ELISA. Data were normalized considering maximalconcentration at the first time point (3 min). t_(1/2)α indicates theinitial plasma half-life; t_(1/2)β indicates the terminal plasmahalf-life; AUC indicates the bioavailability of the tested scDb-IgBD andscFv-IgBD fusion proteins. The moleuclar masses were calculated from theamino acid sequences. Stokes radii (Sr) were determined by sizeexclusion chromatography.

FIG. 9. Immunostimulatory activity of scDb-IgBD fusion proteins. Thebispecific anti-CEA×anti-CD3 scDb-IgBD fusion proteins were analyzed invitro for triggering of IL-2 release from human PBMCs in a targetcell-dependent manner in the absence or presence of human IgG (100μg/ml). CEA-positive target cells (LS1S74T) were grown in microtiterplates and subsequently PBMCs and fusion proteins were added andincubated for 24 h. Subsequently, IL-2 released from activated T cellswas determined by ELISA. Unmodified scDb was included as control.

FIG. 10: SpG_(C3) Mutants. a) SDS-PAGE analysis of scDb (1),scDb-SpG_(C3) (2), scDb-SpG_(C3-Fab) (3), scDb-SpG_(C3-Fc) (4). Gel wasstained with Coomassie brilliant blue G-250. b) Binding ofscDb-SpG_(C3), scDb-SpG_(C3-Fab), scDb-SpG_(C3-Fc) to human IgG, Fab andFc analyzed by ELISA. scDb-SpG_(C3), scDb-SpG_(C3-Fab),scDb-SpG_(C3-Fc), were analyzed for binding to immobilized human serumIgG as well as Fab and Fc fragments thereof. c) Binding of scDb,scDb-SpG_(C3), scDb-SpG_(C3-Fab), scDb-SpG_(C3-Fc) to CEA analyzed byELISA. d) Plasma half-life of scDb-SpG_(C3-Fab): scDb-SpG_(C3-Fab) wasi.v. injected into CD1 mice (25 μg/animal) and serum concentrations ofthe antibody molecules were determined at different time points byELISA. Data were normalized considering maximal concentration at thefirst time point (3 min).

FIG. 11: Production and IgG-binding of an SpG-C3-Diabody-scTRAIL fusionprotein. a) Composition of the fusion protein composed of an N-terminalSpG_(C3) domain, an anti-EGFR diabody and a single-chain derivative ofTRAIL (scTRAIL). b) Detection of purified fusion protein by Westernblotwith anti-TRAIL or anti-FLAG-tag antibodies. A band corresponding to theexpected size of approximately 100 kDa is detected. c) Binding of thefusion protein (10 μg/ml) to immobilized human serum IgG in ELISA. Boundfusion protein was detected with an anti-FLAG-tag antibody and anHRP-conjugated anti-mouse antibody. The fusion protein was omitted inthe control.

FIG. 12: Amino acid sequences of the SpG-C3 binding epitop on the CH1domains of human, mouse, and rat IgG according to EU index as in Kabat.

FIG. 13: Nucleic acid and amino acid sequence of scFv-SpG-C3 fusionprotein (anti-CEA). SpG_(C3) sequence is markes with a grey box, theleader sequence is underlined.

FIG. 14: Nucleic acid and amino acid sequence of scDb-SpG-C3 fusionprotein (anti-CEA×anti-CD3). SpG_(C3) sequence is markes with a greybox, the leader sequence is underlined.

FIG. 15: Nucleic acid and amino acid sequence of SpG-C3-Db-scTRAIL(anti-human EGFR) fusion protein. SpG_(C3) sequence is markes with agrey box, the leader sequence is underlined.

FIG. 16: Pharmakocinetic properties of scDb-SpG_(C3) and scDb-ABD_(H).a) ScDb, scDb-SpG_(C3) and scDb-ABD_(H) fusion proteins were i.v.injected into CD1 mice (25 μg/animal) and serum concentrations atdifferent time points were determined by ELISA. The 3 min value was setto 100% for normalization. b) Plasma concentrations shown for the first1 h. c) AUC determined over the first 24 h. d) Initial plasma half-livesdetermined for the first 3 time points (up to 1 h). Plasma half-livesand AUC_(0-24h) of scDb, scDb-SpG_(C3) and scDb-ABD_(H) were calculatedfrom the serum concentrations by Excel and statistics were performedusing a T-test with GraphPad Prism.

FIG. 17: Comparison of IL-2 release using scDb-SpG_(C3-Fab) andscDb-ABD_(H) fusion proteins. LS174T cells were incubated with varyingconcentrations of scDb (a and c), scDb-ABD_(H) (b) or scDb-SpG_(c3-Fab)(d) before adding human PBMCs. The scDb fusion proteins were eitherpreincubated without (white symbols) or with 1 mg/ml HSA (a, b) or 100μg/ml human IgG (c, d) corresponding approximately to 1/50 of the normalplasma concentrations (black symbols). After 24 h, IL-2 release into thesupernatant was determined by ELISA.

DETAILED DESCRIPTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The term “complex” as used herein, refers to a whole that comprehends anumber of individual components, parts or moieties which are in closeproximity to each other and fulfil a common or interrelated function.The individual parts of such complex may fulfil differing functions inorder to achieve the common function of the complex, i.e. one part ofthe complex may mediate one function (e.g. the binding of the complex)whilst the other part of the complex may mediate a different function(e.g. the activity of the complex) in order to fulfil the commonfunction (e.g. of a site specific activity). The individual moieties ofa complex may be of the same or of differing nature, i.e. they may becomposed of the same, a similar or of differing chemical entities suchas but not limited to nucleotides, amino acids, nucleic acids, peptides,polypeptides, proteins, carbohydrates, and/or lipids. Exemplified, acomplex may comprise a number of associated proteins, or a mixture ofone or more proteins and one or more nucleic acids or a mixture of oneor more proteins and one or more lipids and/or carbohydrates. It isunderstood that any other combination of identical, similar or differingchemical entities is also encompassed. The individual moieties of acomplex may or may not be interconnected. Typically, the individualparts of a complex are connected via covalent or non-covalent bonds.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and are understood as a polymeric or oligomeric macromoleculemade from nucleotide monomers. Nucleotide monomers are composed of anucleobase, a five-carbon sugar (such as but not limited to ribose or2′-deoxyribose), and one to three phosphate groups. Typically, apolynucleotide is formed through phosphodiester bonds between theindividual nucleotide monomers. In the context of the present inventionreferred to nucleic acid molecules include but are not limited toribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixturesthereof such as e.g. RNA-DNA hybrids. The nucleic acids, can e.g. besynthesized chemically, e.g. in accordance with the phosphotriestermethod (see, for example, Uhlmann & Peyman (1990) Chemical Reviews90:543-584). “Aptamers” are nucleic acids which bind with high affinityto a polypeptide. Aptamers can be isolated by selection methods such asSELEmir146-a (see e.g. Jayasena (1999) Clin. Chem. 45:1628-50; Klug andFamulok (1994) M. Mol. Biol. Rep. 20:97-107; U.S. Pat. No. 5,582,981)from a large pool of different single-stranded RNA molecules. Aptamerscan also be synthesized and selected in their mirror-image form, forexample as the L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol.14:1116-1119; Klussmann et al. (1996) Nat. Biotechnol. 14:1112-1115).Forms which have been isolated in this way enjoy the advantage that theyare not degraded by naturally occurring ribonucleases and, therefore,possess greater stability.

The terms “protein” and “polypeptide” are used interchangeably hereinand refer to any peptide-bond-linked chain of amino acids, regardless oflength or post-translational modification. Proteins usable in thepresent invention (including protein derivatives, protein variants,protein fragments, protein segments, protein epitops and proteindomains) can be further modified by chemical modification. This meanssuch a chemically modified polypeptide comprises other chemical groupsthan the 20 naturally occurring amino acids. Examples of such otherchemical groups include without limitation glycosylated amino acids andphosphorylated amino acids. Chemical modifications of a polypeptide mayprovide advantageous properties as compared to the parent polypeptide,e.g. one or more of enhanced stability, increased biological half-life,or increased water solubility.

As used herein, the term “variant” is to be understood as apolynucleotide or protein which differs in comparison to thepolynucleotide or protein from which it is derived by one or morechanges in its length or sequence. The polypeptide or polynucleotidefrom which a protein or nucleic acid variant is derived is also known asthe parent or parental polypeptide or polynucleotide. The term “variant”comprises “fragments” or “derivatives” of the parent molecule.Typically, “fragments” are smaller in length or size than the parentmolecule, whilst “derivatives” exhibit one or more differences in theirsequence in comparison to the parent molecule. Also encompassed aremodified molecules such as but not limited to post-translationallymodified proteins (e.g. glycosylated, biotinylated, phosphorylated,ubiquitinated, palmitoylated, or proteolytically cleaved proteins) andmodified nucleic acids such as methylated DNA. Also mixtures ofdifferent molecules such as but not limited to RNA-DNA hybrids, areencompassed by the term “variant”. Typically, a variant is constructedartificially, preferably by gene-technological means whilst the parentpolypeptide or polynucleotide is a wild-type protein or polynucleotide.However, also naturally occurring variants are to be understood to beencompassed by the term “variant” as used herein. Further, the variantsusable in the present invention may also be derived from homologs,orthologs, or paralogs of the parent molecule or from artificiallyconstructed variant, provided that the variant exhibits at least onebiological activity of the parent molecule, i.e. is functionally active.

The changes in the nucleotide or amino acid sequence may be nucleotideor amino acid exchanges, insertions, deletions, 5′- or 3′ truncations,N- or C-terminal truncations, or any combination of these changes, whichmay occur at one or several sites. In preferred embodiments, a variantusable in the present invention exhibits a total number of up to 200 (upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, or 200) changes in the nucleotide or amino acid sequence (i.e.exchanges, insertions, deletions, and/or truncations). Amino acidexchanges may be conservative and/or non-conservative. Alternatively oradditionally, a “variant” as used herein, can be characterized by acertain degree of sequence identity to the parent polypeptide or parentpolynucleotide from which it is derived. More precisely, a proteinvariant in the context of the present invention exhibits at least 70%sequence identity to its parent polypeptide. A polynucleotide variant inthe context of the present invention exhibits at least 70% sequenceidentity to its parent polynucleotide. Preferably, the sequence identityof protein variants is over a continuous stretch of 20, 30, 40, 45, 50,60, 70, 80, 90, 100 or more amino acids. Preferably, the sequenceidentity of polynucleotide variants is over a continuous stretch of 60,90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides.

The term “at least 70% sequence identity” is used throughout thespecification with regard to polypeptide and polynucleotide sequencecomparisons. This expression preferably refers to a sequence identity ofat least 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99% tothe respective reference polypeptide or to the respective referencepolynucleotide.

In case where two sequences are compared and the reference sequence isnot specified in comparison to which the sequence identity percentage isto be calculated, the sequence identity is to be calculated withreference to the longer of the two sequences to be compared, if notspecifically indicated otherwise. If the reference sequence isindicated, the sequence identity is determined on the basis of the fulllength of the reference sequence indicated by SEQ ID, if notspecifically indicated otherwise. For example, a peptide sequenceconsisting of 358 amino acids compared to the amino acid sequence of anIgG molecule may exhibit a maximum sequence identity percentage of80.09% (358/447) while a sequence with a length of 224 amino acids mayexhibit a maximum sequence identity percentage of 50.11% (224/447). Thesimilarity of nucleotide and amino acid sequences, i.e. the percentageof sequence identity, can be determined via sequence alignments. Suchalignments can be carried out with several art-known algorithms,preferably with the mathematical algorithm of Karlin and Altschul(Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), withhmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTALalgorithm (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680)available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or onhttp://www.ebi.ac.uk/Tools/clustalw2/index.html or onhttp://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html.Preferred parameters used are the default parameters as they are set onhttp://www.ebi.ac.uk/Tools/clustalw/ orhttp://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410.BLAST polynucleotide searches are performed with the BLASTN program,score=100, word length=12. BLAST protein searches are performed with theBLASTP program, score=50, word length=3. To obtain gapped alignments forcomparative purposes, Gapped BLAST is utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programsare used. Sequence matching analysis may be supplemented by establishedhomology mapping techniques like Shuffle-LAGAN (Brudno M. (2003b)Bioinformatics 19 Suppl 1:I54-I62) or Markov random fields. Whenpercentages of sequence identity are referred to in the presentapplication, these percentages are calculated in relation to the fulllength of the longer sequence, if not specifically indicated otherwise.“Hybridization” can also be used as a measure of sequence identity orhomology between two nucleic acid sequences. A nucleic acid sequenceencoding F, N, or M2-1, or a portion of any of these can be used as ahybridization probe according to standard hybridization techniques.Hybridization conditions are known to those skilled in the art and canbe found, for example, in Current Protocols in Molecular Biology, JohnWiley & Sons, N. Y., 6.3.1-6.3.6, 1991. “Moderate hybridizationconditions” are defined as equivalent to hybridization in 2× sodiumchloride/sodium citrate (SSC) at 30° C., followed by a wash in 1×SSC,0.1% SDS at 50° C. “Highly stringent conditions” are defined asequivalent to hybridization in 6× sodium chloride/sodium citrate (SSC)at 45° C., followed by a wash in 0.2×SSC, 0.1% SDS at 65° C.

The term “immunoglobulin (Ig)” as used herein refers to immunityconferring glycoproteins of the immunoglobulin superfamily. “Surfaceimmunoglobulins” are attached to the membrane of effector cells by theirtransmembrane region and encompass molecules such as but not limited toB-cell receptors, T-cell receptors, class I and II majorhistocompatibility complex (MHC) proteins, beta-2 microglobulin (β2M),CD3, CD4 and CD8. Typically, the term “antibody” as used herein refersto secreted immunoglobulins which lack the transmembrane region and canthus, be released into the bloodstream and body cavities. Humanantibodies are grouped into different isotypes based on the heavy chainthey possess. There are five types of human Ig heavy chains denoted bythe Greek letters: α, δ, δ, γ, and μ. The type of heavy chain presentdefines the class of antibody, i.e. these chains are found in IgA, IgD,IgE, IgG, and IgM antibodies, respectively, each performing differentroles, and directing the appropriate immune response against differenttypes of antigens. Distinct heavy chains differ in size and composition;α and γ comprise approximately 450 amino acids, while μ and ε haveapproximately 550 amino acids (Janeway et al. (2001) Immunobiology,Garland Science). IgA is found in mucosal areas, such as the gut,respiratory tract and urogenital tract, as well as in saliva, tears, andbreast milk and prevents colonization by pathogens (Underdown & Schiff(1986) Annu. Rev. Immunol. 4:389-417). IgD mainly functions as anantigen receptor on B cells that have not been exposed to antigens andis involved in activating basophils and mast cells to produceantimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437;Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved inallergic reactions via its binding to allergens triggering the releaseof histamine from mast cells and basophils. IgE is also involved inprotecting against parasitic worms (Pier et al. (2004) Immunology,Infection, and Immunity, ASM Press). IgG provides the majority ofantibody-based immunity against invading pathogens and is the onlyantibody isotype capable of crossing the placenta to give passiveimmunity to fetus (Pier et al. (2004) Immunology, Infection, andImmunity, ASM Press). In humans there are four different IgG subclasses(IgG1, 2, 3, and 4), named in order of their abundance in serum withIgG1 being the most abundant (˜66%), followed by IgG2 (˜23%), IgG3 (˜7%)and IgG (˜4%). The biological profile of the different IgG classes isdetermined by the structure of the respective hinge region. IgM isexpressed on the surface of B cells in a monomeric form and in asecreted pentameric form with very high avidity. IgM is involved ineliminating pathogens in the early stages of B cell mediated (humoral)immunity before sufficient IgG is produced (Geisberger et al. (2006)Immunology 118:429-437).

Antibodies are not only found as monomers but are also known to formdimers of two Ig units (e.g. IgA), tetramers of four Ig units (e.g. IgMof teleost fish), or pentamers of five Ig units (e.g. mammalian IgM).Antibodies are typically made of four polypeptide chains comprising twoidentical heavy chains and identical two light chains which areconnected via disulfide bonds and resemble a “Y”-shaped macro-molecule.Each of the chains comprises a number of immunoglobulin domains out ofwhich some are constant domains and others are variable domains.Immunoglobulin domains consist of a 2-layer sandwich of between 7 and 9antiparallel β-strands arranged in two β-sheets. Typically, the heavychain of an antibody comprises four Ig domains with three of them beingconstant (C_(H) domains: C_(H)1, C_(H)2, C_(H)3) domains and one of thebeing a variable domain (V_(H)). The light chain typically comprises oneconstant Ig domain (C_(L)) and one variable Ig domain (V_(L)).Exemplified, the human IgG heavy chain is composed of four Ig domainslinked from N- to C-terminus in the order V_(H)-C_(H)1-C_(H)2-C_(H)3(also referred to as V_(H)-Cγ1-Cγ2-Cγ3), whereas the human IgG lightchain is composed of two immunoglobulin domains linked from N- toC-terminus in the order V_(L)-C_(L), being either of the kappa or lambdatype (Vκ-Cκ or Vλ-Cλ).

Exemplified, the constant chain of human IgG comprises 447 amino acids.Throughout the present specification and claims, the numbering of theamino acid positions in an immunoglobulin are that of the “EU index” asin Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller,C., (1991) Sequences of proteins of immunological interest, 5th ed. U.S.Department of Health and Human Service, National Institutes of Health,Bethesda, Md. The “EU index as in Kabat” refers to the residue numberingof the human IgG1EU antibody. Accordingly, C_(H) domains in the contextof IgG are as follows: “C_(H)1” refers to amino acid positions 118-220according to the EU index as in Kabat; “C_(H)2” refers to amino acidpositions 237-340 according to the EU index as in Kabat; and “C_(H)3”refers to amino acid positions 341-447 according to the EU index as inKabat.

Papain digestion of antibodies produces two identical antigen bindingfragments, called “Fab fragments” (also referred to as “Fab portion” or“Fab region”) each with a single antigen binding site, and a residual“Fc fragment” (also referred to as “Fc portion” or “Fc region”) whosename reflects its ability to crystallize readily. The crystal structureof the human IgG Fc region has been determined (Deisenhofer (1981)Biochemistry 20:2361-2370). In IgG, IgA and IgD isotypes, the Fc regionis composed of two identical protein fragments, derived from the C_(H)2and C_(H)3 domains of the antibody's two heavy chains; in IgM and IgEisotypes, the Fc regions contain three heavy chain constant domains(C_(H)2-4) in each polypeptide chain. In addition, smallerimmunoglobulin molecules exist naturally or have been constructedartificially. The term “Fab′ fragment” refers to a Fab fragmentadditionally comprise the hinge region of an Ig molecule whilst “F(ab′)₂fragments” are understood to comprise two Fab′ fragments being eitherchemically linked or connected via a disulfide bond. Whilst “singledomain antibodies (sdAb)” (Desmyter et al. (1996) Nat. Structure Biol.3:803-811) and “Nanobodies” only comprise a single V_(H) domain, “singlechain Fv (scFv)” fragments comprise the heavy chain variable domainjoined via a short linker peptide to the light chain variable domain(Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883).Divalent single-chain variable fragments (di-scFvs) can be engineered bylinking two scFvs (scFvA-scFvB). This can be done by producing a singlepeptide chain with two V_(H) and two V_(L) regions, yielding “tandemscFvs” (V_(H)A-V_(L)A-V_(H)B-V_(L)B). Another possibility is thecreation of scFvs with linkers that are too short for the two variableregions to fold together, forcing scFvs to dimerize. Usually linkerswith a length of 5 residues are used to generate these dimers. This typeis known as “diabodies”. Still shorter linkers (one or two amino acids)between a V_(H) and V_(L) domain lead to the formation of monospecifictrimers, so-called “triabodies” or “tribodies”. Bispecific diabodies areformed by expressing to chains with the arrangement V_(H)A-V_(L)B andV_(H)B-V_(L)A or V_(L)A-V_(H)B and V_(L)B-V_(H)A, respectively.Single-chain diabodies (scDb) comprise a V_(H)A-V_(L)B and aV_(H)B-V_(L)A fragment which are linked by a linker peptide (P) of 12-20amino acids, preferably 14 amino acids, (V_(H)A-V_(L)B-P-V_(H)B-V_(L)A).“Bi-specific T-cell engagers (BiTEs)” are fusion proteins consisting oftwo scFvs of different antibodies wherein one of the scFvs binds to Tcells via the CD3 receptor, and the other to a tumor cell via a tumorspecific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244).Dual affinity retargeting molecules (“DART” molecules) are diabodiesadditionally stabilized through a C-terminal disulfide bridge.

The term “immunoglobulin (Ig) binding moiety” as used herein refers to amoiety or part of a complex which interacts with an immunoglobulin.Typically an Ig binding moiety comprises a polypeptide or a proteinwhich binds to the heavy and/or to the light chain of an Ig, preferablyan antibody. An Ig binding moiety may comprise an “Ig binding domain(IgBD)” as well as further domains fulfilling additional functions suchas but not limited to stabilizing the Ig binding moiety, or promotingthe Ig binding potential of the IgBD.

The term “Ig binding domain (IgBD)” as used herein refers to domainswhich mediate the actual binding of the Ig binding moiety to the Igmolecule. An IgBD may bind to any of the domains of an Ig molecule, i.e.to the variable domains V_(H) or V_(L) and/or to the constant domainsC_(H)1, C_(H)2, C_(H)3 and/or C_(L) of an Ig molecule. Typically an IgBDhas an affinity to bind to an Ig molecule at neutral pH (i.e. pH 7),however also binding at lower of higher pH values, e.g. at pH values 5,6, or 8, may occur. The affinity of an IgBD to bind to Ig molecules maylie below 10⁻⁶ M, often below 10⁻⁷ M, or even below 10⁻⁸ M. Typically,IgBDs are derived from Ig binding proteins of gram-positive bacteria.These include but are not limited to Protein A from Staphylococcusaureus, streptococcal Protein G, and Protein L from Peptostreptococcusmagnus (now: Finegoldia magna).

“Protein A (SpA)” is a 40-60 kDa surface protein originally found in thecell wall of Staphylococcus aureus. It binds immunoglobulins, mostnotably IgGs, from many mammalian species through an interaction of twoα-helices of its IgBDs (A, B, C, D, E) with the C_(H)2 and C_(H)3domains in the Fc fragment of an Ig molecule. Protein A binds with highaffinity to human IgG1 and IgG2 as well as mouse IgG2a and IgG2b butonly with moderate affinity to human IgM, IgA and IgE as well as tomouse IgG3 and IgG1.

“Protein G (SpG)” is an immunoglobulin-binding protein expressed ingroup C and G streptococcal strains which is similar to Protein A butexhibits different specificities. It is a cell surface protein of about65-kDa that binds to the Fc region of IgG molecules (in particular toIgG1, IgG2 or IgG4) as well as to serum albumin. The amino acidsequences of the individual IgBDs are identical from streptococcalstrains G148, GX7805, and GX7809 (Guss et al. (1986) EMBO J.5:1567-1575). Protein G consists of repetitively arranged domains withthe C-terminal domains (C1, C2, C3, also referred to as domains B1-B3)being responsible for IgG binding and the domains in the N-terminal halfof the protein (domains A1, A2, A3) binding to serum albumin. The singleIgBDs of Protein G show a common secondary structure consisting of acentral α-helix packed against a four-stranded,antiparallel-parallel-antiparallel β-sheet. The amino acid sequences ofdomains C1 and C2 are 90% and 93%, respectively, identical to thesequence of domain C3. The amino acid sequences of Ig-binding domainsC1, C2, and C3 of streptococcal Protein G are as followed:

SpG-C1: (SEQ ID NO: 16)TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKT FTVTE SpG-C2:(SEQ ID NO: 17) TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKT FTVTESpG-C3: (SEQ ID NO: 1)TYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKT FTVTE

Whilst in most streptococcal strains Protein G comprises all threeIg-binding domains (C1-C3), some stains contain a Protein G onlycomprising two Ig-binding domains. Exemplified, streptococcal strainGX7809 contains a Protein G with only two Ig-binding domains, whereinthe first domain is identical to C1 of G148 and GX7805 and the lastdomain identical to C3 of G148 and GX7805.

The interaction between the IgBD of Protein G, in particular the C1 andC2 domains of SpG, and the Fc fragment of an immunoglobulin is mediatedby the α-helix and the third β-strand within the IgBD (Gronenborn &Clore (1993) J. Mol. Biol. 233:331-335). The C3 domain interacts withthe Fab fragment of an Ig molecule by binding to the surface exposedregion of the C_(H)1 domain of the Ig molecule. This interaction of theC3 domain with the Fab fragment is mediated through an antiparallelalignment of the second β-strand from domain C3 with the seventhβ-strand from the C_(H)1 domain of the Ig molecule which affects theextension of the four stranded β-sheet of domain C3 into the C_(H)1domain (Derrick & Wigley (1994) J. Mol. Biol. 243:906-918). Morespecifically, the C3 domain interacts with the amino acid positions122-127 and/or 207-214 of the C_(H)1 domain according to the EU index asin Kabat (see FIG. 1).

Unlike Protein A and Protein G, which bind to the heavy chain ofimmunoglobulins, “Protein L (PpL)” from Peptostreptococcus magnus bindsthrough light chain interactions to those Ig molecules that containkappa light chains. In the process PpL does not interfere with theantigen-binding site of the Ig molecule. Protein L binds torepresentatives of all antibody classes, including IgG, IgM, IgA, IgEand IgD as well as to scFv and Fab fragments.

The availability of a substance (for example a metabolite, drug,signaling molecule, radioactive nuclide, or other substance) in the bodyis dependent on several factors, such as its concentration in the bloodplasma and the speed of its clearance from the body. The overallpersistence of a substance in the body, i.e. the length of time asubstance spends in the body, is expressed as the “mean resistance time(MRT)”. The MRT depends on various factors such as the individual's bodysize, the rate at which the substance moves through and react within thebody, and if applicable the amount of a substance, e.g. apharmaceutical, administered. The MRT is also dependent on the overallability of the body to eliminate a certain substance, e.g. a drug, fromthe plasma. In mammals, plasma clearance is achieved by the mainclearing organs: the kidney and the liver. The term “plasma clearance”as used herein thus, refers to the volume of plasma that is cleared of acertain substance in a given time and is measured in units of avolumetric flow rate (volume/time).

The time it takes for the blood concentration of a substance to fall byone half is refered to as the “plasma half-life” or the “serumhalf-life” of a substance, irrespective of the factors (e.g. plasmaclearance, absorbance by the tissue) which cause the decrease inconcentration. The term “plasma” refers to the complete soluble fractionof the blood, whilst the term “serum” refers to plasma devoid ofcoagulation factors, i.e. obtained after coagulation of blood. Both, theplasma half-life and the serum half-life are measured as concentrationin the blood.

A differentiation may be made between the initial and the terminalplasma or serum half-life of a substance. The terms “initial plasmahalf-life” (or “initial serum half-life”) and “distribution plasmahalf-life” (or “distribution serum half-life”) are used interchangeablyherein and are abbreviated as t_(1/2)α. The initial plasma half-liferefers to the phase of rapid drug disappearance from the blood whichoccurs immediately after the dose is given, and which may lead to a verysubstantial decrement in drug concentrations in the blood. This initialphase of rapid drug disappearance is determined mainly by reversibledistribution of drug out of the “central” compartment, of which thevascular system is a component, into storage sites in peripheraltissues; very little of this initial rapid decline is determined byelimination or clearance. Typically, the initial phase lasts from a fewminutes (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min) to a few hours (i.e.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours).

The terms “terminal plasma half-life” (or “terminal serum half-life”) or“elimination plasma half-life” (or “elimination serum half-life”) areused interchangeably herein and are abbreviated as t_(1/2)β. Theelimination plasma half-life is generally determined only after drugdistribution equilibrium has been attained, i.e. after the distributionof the administered substance in the various body tissues is complete.The blood concentration curve enters a less rapid phase of drugdisappearance, termed the elimination phase, during which drugdisappearance is determined mainly by irreversible clearance.Accordingly, the terminal plasma half-life (t_(1/2)β) is determined byclearance (CL) and volume of distribution (V_(D)) and the relationshipis described by the following equation:

$t_{1/2} = \frac{\ln \; {2 \cdot V_{D}}}{C\; L}$

Depending on the substance in question, the relationship between theinitial plasma half-life and the terminal plasma half-life of suchsubstance may be complex, taking into account factors including itsaccumulation in the tissues and receptor interactions (Toutain &Bousquet-Melou (2004) J. Vet. Pharmacol. Therap. 27:427-439). Both, theinitial plasma half-life (or “initial serum half-life”) and the terminalplasma half-life (or “terminal serum half-life”) of a substance, e.g. apharmaceutical, can be influenced in order to extend the bioavailabilityof such substance in the body. The bioavailability of a substance may bedetermined by measuring the concentration of said substance in the blood(plasma or serum) at certain time intervals after administration andestablishing the area under the concentration-time-curve. The value ofthe “Area under the curve (AUC)” is proportional to the amount of thesubstance being available in the bloodstream.

Exemplified, the reduction of plasma clearance, e.g. by increasing thehydrodynamic volume of such substance to reduce renal clearance or byutilizing recycling processes via the FcRn, may lead to a prolongedterminal plasma half-life of the respective substance and thereby to anincreased bioavailability in the body.

In the context of the present application it is preferred that the serumhalf-life, preferably the terminal serum half-life, of apharmaceutically active moiety can be prolonged by reducing its plasmaclearance and allowing for its recycling via the FcRn by complexing thepharmaceutically active moiety to an IgBD, preferably the C3 IgBD ofstreptococcal Protein G as described in detail above. The terms“prolonged” and “extended” or “prolongation” and “extension” are usedinterchangeably herein referring to an increase in the length of time,preferably in the lengths of the serum half-life, in particular theinitial and/or terminal serum half-life.

The term “pharmaceutically active moiety” as used herein, is understoodto refer to a part or moiety of a complex which mediates apharmaceutical effect including but not limited to prophylactic,therapeutic, and/or diagnostic effects.

As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis”of a disease or disorder means preventing that such disease or disorderoccurs in a patient. Accordingly, a moiety having a prophylactic effectprevents the onset of a disease or disorder in a patient.

As used herein, “treat”, “treating”, “treatment” or “therapy” of adisease or disorder means accomplishing one or more of the following:(a) reducing the severity of the disorder; (b) limiting or preventingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) inhibiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting or preventing recurrence of the disorder(s)in an individual that has previously had the disorder(s); and (e)limiting or preventing recurrence of symptoms in individuals that werepreviously symptomatic for the disorder(s). Accordingly, a moiety havinga therapeutic effect treats the symptoms of a disease or disorder byaccomplishing one or more of above named effects (a)-(e).

The terms “identify”, “identifying”, “identification” or “diagnosis” ofa disease or disorder are used herein to refer to the determination ofthe nature and the cause of a disease or disorder. Accordingly, a moietyhaving a diagnostic effect allows for the determination of the natureand the cause of a disease or disorder.

“Symptoms” of a disease or disorder are implication of the disease ordisorder noticeable by the tissue, organ or organism having such diseaseor disorder and include but are not limited to pain, weakness,tenderness, strain, stiffness, and spasm of the tissue, an organ or anindividual as well as the presence, absence, increase, decrease, ofspecific indicators such as biomarkers or molecular markers. The term“disease” and “disorder” as used herein, refer to an abnormal condition,especially an abnormal medical condition such as an illness or injury,wherein a tissue, an organ or an individual is not able to efficientlyfulfil its function anymore. Typically, but not necessarily, a diseaseor disorder is associated with specific symptoms or signs indicating thepresence of such disease or disorder.

A pharmaceutically active moiety typically comprises a biological and/orchemical pharmaceutical. “Chemical pharmaceuticals” are typicallyunderstood to refer to chemical compounds synthesized artificially whichare effective in the prevention, treatment or diagnosis of disorders ordiseases. “Biologicals” are typically understood to refer to medicaldrugs produced using biotechnological means and are used forprophylactic, therapeutic, and/or in vivo diagnostic purposes.Biologicals include but are not limited to peptides, polypeptides,proteins and nucleic acids (e.g. DNA, RNA, or hybrids thereof). Approvedtherapeutic biologicals include but are not limited to hormones (e.g.insulin, hGH, FSH, Glucagon-like peptide 1, parathyroid hormone,calcitonin, lutropin, glucagon), growth factors (e.g. erythropoietin,G-CSF/GM-CSF, IGF-1), interferons (e.g. IFN-α, IFN-β, IFN-γ),interleukins (e.g. IL-2, IL-11, IL-1Ra), coagulation factors (e.g.factor VIII, factor IX, factor VIIa, thrombin), thrombolytics andanti-coagulants (e.g. t-PA, hirudin, activated protein C), enzymes (e.g.α-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase,urate oxidase, DNase), antigen-binding molecule such as antibodies andantibody fragments (e.g. IgG, Fab), and fusion proteins thereof (e.g.TNFR2-Fc, TMP-Fc, CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

A “peptide linker” (or short: “linker”) in the context of the presentinvention refers to an amino acid sequence which sterically separatestwo parts or moieties of a complex, e.g. two peptides or proteins.Typically such linker consists of between 1 and 100 amino acids having aminimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acids, and a maximum length of at least 100, 95, 90, 85, 80, 75,70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. Theindicated preferred minimum and maximum lengths of the peptide linkeraccording to the present invention may be combined, if such acombination makes mathematically sense, e.g. such linker may consist of1-15, or 12-40, or 25-75, or 1-100 amino acids. Peptide linkers may alsoprovide flexibility among the two moieties that are linked together.Such flexibility is generally increased if the amino acids are small.Accordingly, flexible peptide linkers comprise an increased content ofsmall amino acids, in particular of glycins and/or alanines, and/orhydrophilic amino acids such as serines, threonines, asparagines andglutamines. Preferably, more than 20%, 30%, 40%, 50%, 60% or more of theamino acids of the peptide linker are small amino acids.

The term “cleavage site” as used herein refers to an amino acid sequenceor nucleotide sequence wherein this sequence directs the division of acomplex or a macromolecule (e.g. a nucleic acid or a protein), e.g.because it is recognized by a cleaving enzyme, and/or can be divided.Typically, a polypeptide chain is cleaved by hydrolysis of one or morepeptide bonds that link the amino acids and a polynucleotide chain iscleaved by hydrolysis of one or more of the phosphodiester bond betweenthe nucleotides. Cleavage of peptide- or phosphodiester-bonds mayoriginate from chemical or enzymatic cleavage. Enzymatic cleavage refersto such cleavage being attained by proteolytic enzymes including but notlimited to restriction endonuclease (e.g. type I, type II, type II, typeIV or artificial restriction enzymes) and endo- or exo-peptidases or-proteases (e.g. serine-proteases, cysteine-proteases,metallo-proteases, threonine proteases, aspartate proteases, glutamicacid proteases). Typically, enzymatic cleavage occurs due toself-cleavage or is affected by an independent proteolytic enzyme.Enzymatic cleavage of a protein or polypeptide can happen either co- orpost-translational. Accordingly, the term “endopeptidase cleavage site”used herein, refers to a cleavage cite within the amino acid ornucleotide sequence where this sequence is cleaved or is cleavable by anendopeptidase (e.g. trypsin, pepsin, elastase, thrombin, collagenase,furin, thermolysin, endopeptidase V8, cathepsins).

The term “self-cleavage site” as used herein refers to a cleavage sitewithin the amino acid sequence where this sequence is cleaved or iscleavable without such cleavage involving any additional molecule. It isunderstood that cleavage sites typically comprise several amino acids.Thus, the cleavage site may also serve the purpose of a peptide linker,i.e. sterically separating two peptides or proteins.

As used herein, the term “vector” refers to a protein or apolynucleotide or a mixture thereof which is capable of being introducedor of introducing the proteins and/or nucleic acid comprised thereininto a cell. In the context of the present invention it is preferredthat the genes of interest encoded by the introduced polynucleotide areexpressed within the cell upon introduction of the vector or vectors.Examples of suitable vectors include but are not limited to plasmids,cosmids, phages, viruses or artificial chromosomes.

The terms “pharmaceutical”, “medicament” and “drug” are usedinterchangeably herein, referring to a substance and/or a combination ofsubstances being used for the identification, prevention or treatment ofa disease or disorder.

The terms “preparation” and “composition” are intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with the active compound.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “active ingredient” refers to the substance in a pharmaceuticalcomposition or formulation that is biologically active, i.e. thatprovides pharmaceutical value. A pharmaceutical composition may compriseone or more active ingredients which may act in conjunction with orindependently of each other. The active ingredient can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as but not limited to those derivedfrom hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as but not limited to thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

The term “carrier”, as used herein, refers to a pharmacologicallyinactive substance such as but not limited to a diluent, excipient,surfactants, stabilizers, physiological buffer solutions or vehicleswith which the therapeutically active ingredient is administered. Suchpharmaceutical carriers can be liquid or solid. Liquid carrier includebut are not limited to sterile liquids, such as saline solutions inwater and oils, including but not limited to those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. A saline solution is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Examplesof suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

Suitable pharmaceutical “excipients” include starch, glucose, lactose,sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like.

“Surfactants” include anionic, cationic, and non-ionic surfactants suchas but not limited to sodium deoxycholate, sodium dodecylsulfate, TritonX-100, and polysorbates such as polysorbate 20, polysorbate 40,polysorbate 60, polysorbate 65 and polysorbate 80.

“Stabilizers” include but are not limited to mannitol, sucrose,trehalose, albumin, as well as protease and/or nuclease antagonists.

“Physiological buffer solution” include but are not limited to sodiumchloride solution, demineralized water, as well as suitable organic orinorganic buffer solutions such as but not limited to phosphate buffer,citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPESbuffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPSbuffer (3 morpholino-1 propanesulphonic acid). The choice of therespective buffer in general depends on the desired buffer molarity.Phosphate buffer are suitable, for example, for injection and infusionsolutions.

The term “adjuvant” refers to agents that augment, stimulate, activate,potentiate, or modulate the immune response to the active ingredient ofthe composition at either the cellular or humoral level, e.g.immunologic adjuvants stimulate the response of the immune system to theactual antigen, but have no immunological effect themselves. Examples ofsuch adjuvants include but are not limited to inorganic adjuvants (e.g.inorganic metal salts such as aluminium phosphate or aluminiumhydroxide), organic adjuvants (e.g. saponins or squalene), oil-basedadjuvants (e.g. Freund's complete adjuvant and Freund's incompleteadjuvant), cytokines (e.g. IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, andINF-γ) particulate adjuvants (e.g. immuno-stimulatory complexes(ISCOMS), liposomes, or biodegradable microspheres), virosomes,bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides),synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptideanalogues, or synthetic lipid A), or synthetic polynucleotides adjuvants(e.g polyarginine or polylysine).

An “effective amount” or “therapeutically effective amount” is an amountof a therapeutic agent sufficient to achieve the intended purpose. Theeffective amount of a given therapeutic agent will vary with factorssuch as the nature of the agent, the route of administration, the sizeand species of the animal to receive the therapeutic agent, and thepurpose of the administration. The effective amount in each individualcase may be determined empirically by a skilled artisan according toestablished methods in the art.

EMBODIMENTS

In a first aspect the present invention relates to a complex comprising(i) an immunoglobulin (Ig) binding moiety and (ii) a pharmaceuticallyactive moiety, wherein the Ig binding moiety specifically binds to theconstant domain 1 of the heavy chain (C_(H)1) of an Ig molecule.

In preferred embodiments, the Ig binding moiety prolongs the serumhalf-life of the pharmaceutically active moiety, i.e. thepharmaceutically active moiety exhibits a prolonged serum half-life whenbeing part of the complex of the first aspect of the invention. Inpreferred embodiments the complex of the first aspect is thus, used forextending the serum half-life, preferably the serum half-life of thepharmaceutically active moiety. It is particularly preferred that theinitial and/or the terminal serum half-life are extended. It is furtherpreferred that the bioavailabilty, more preferably the bioavailabilty ofthe pharmaceutically active moiety, is increased.

In the context of the present invention it is preferred that the Igbinding moiety binds to mammalian, avian, fish or reptile Ig, inparticular to Igs of laboratory animals including but not limited tomouse, rat and rabbit, and/or domestic animals including but not limitedto guinea pig, rabbit, horse, donkey, camel, cow, sheep, goat, pig,chicken, duck, goose, parrot, canary bird, cat, dog, goldfish, trout,pangasius, carp, koi, perch, catfish, salmon, turtle, tortoise, snake,and lizard, and/or primates including but not limited to gibbons,lemurs, chimpanzees, bonobos, gorillas, and human beings. It isparticularly preferred that the Ig binding moiety binds to the Ig ofhuman beings. Preferably, the binding of the Ig binding moiety to the Igmolecule occurs in vivo, i.e. within the body of a mammal, bird, fish,or reptile, in particular within the body of a mammal, bird, fish, orreptile as specified above. Accordingly, preferably, the Ig bindingmoiety binds to an Ig molecule in vivo.

In further embodiments of the present invention, the Ig binding moietybinds to IgA, IgD, IgE, IgG, and/or IgM, preferably to an IgG ofsubclasses IgG1, IgG2, IgG3, and/or IgG4, more preferably to IgG1, IgG2,and/or IgG4.

The Ig binding moiety preferably binds to the Fab fragment and/or the Fcportion of an immunoglobulin molecule. It is particularly preferred thatthe Ig binding moiety binds to the Fab portion of an Ig molecule, andthat it optionally also binds to the Fc fragment. Thus, in preferredembodiments, the Ig binding moiety has a structure allowing for thebinding to both, the Fc portion and the Fab portion of an immunoglobulinmolecule (e.g. as illustrated in FIG. 1 b). In further preferredembodiments the Ig binding moiety has a structure allowing for thebinding to either the Fc portion or the Fab portion of an immunoglobulinmolecule (e.g. as illustrated in FIG. 1 c for a Fab-binding Ig bindingmoiety). Preferably, the ability to bind to only one of the Fc portionor the Fab portion of an immunoglobulin molecule is due to thefunctional inactivation (e.g. the structural deletion of all or parts ofthe Fab- or Fc-binding site, or the functional inactivation via aminoacid deletions, replacements, additions, mutations or exchanges) of therespective binding site of Ig binding moiety via genetic engineering. Inpreferred embodiments, the Ig binding moiety has an affinity to bind toan Ig molecule at neutral pH (i.e. pH 7), preferably with an affinity ofbelow 10⁻⁶ to below 10⁻⁹ M, i.e. with an affinity of below 10⁻⁶, below10⁻⁷ M, below 10⁻⁸ M, or below 10⁻⁹ M. It is particularly preferred thatthe Ig binding moiety binds to the Fab fragment of an Ig molecule withan affinity of 10⁻⁷ M to 10⁻⁶ M (i.e. with an affinity of 1×10⁻⁷ M,1.1×10⁻⁷ M, 1.2×10⁻⁷ M, 1.3×10⁻⁷ M, 1.4×10⁻⁷ M, 1.5×10⁻⁷ M, 1.6 ×10⁻⁷ M,1.7×10⁻⁷ M, 1.8×10⁻⁷ M, 1.9×10⁻⁷ M, 2×10⁻⁷ M, 2.1×10⁻⁷ M, 2.2×10⁻⁷ M,2.3×10⁻⁷ M, 2.4×10⁻⁷ M, 2.5×10⁻⁷ M, 2.6×10⁻⁷ M, 2.7×10⁻⁷ M, 2.8×10⁻⁷ M,2.9×10⁻⁷ M, 3×10⁻⁷ M, 3.1×10⁻⁷ M, 3.2×10⁻⁷ M, 3.3×10⁻⁷ M, 3.4×10⁻⁷ M,3.5×10⁻⁷ M, 3.6×10⁻⁷ M, 3.7×10⁻⁷ M, 3.8×10⁻⁷ M, 3.9×10⁻⁷ M, 4×10⁻⁷ M,4.1×10⁻⁷ M, 4.2×10⁻⁷ M, 4.3×10⁻⁷ M, 4.4×10⁻⁷ M, 4.5×10⁻⁷ M, 4.6×10⁻⁷ M,4.7×10⁻⁷ M, 4.8×10⁻⁷ M, 4.9×10⁻⁷ M, 5×10⁻⁷ M, 5.1×10⁻⁷ M, 5.2×10⁻⁷ M,5.3×10⁻⁷ M, 5.4×10⁻⁷ M, 5.5×10⁻⁷ M, 5.6×10⁻⁷ M, 5.7×10⁻⁷ M, 5.8×10⁻⁷ M,5.9×10⁻⁷ M, 6×10⁻⁷ M, 6.1×10⁻⁷ M, 6.2×10⁻⁷ M, 6.3×10⁻⁷ M, 6.4×10⁻⁷ M,6.5×10⁻⁷ M, 6.6×10⁻⁷ M, 6.7×10⁻⁷ M, 6.8×10⁻⁷ M, 6.9×10⁻⁷ M, 7×10⁻⁷ M,7.1×10⁻⁷ M, 7.2×10⁻⁷ M, 7.3×10⁻⁷ M, 7.4×10⁻⁷ M, 7.5×10⁻⁷ M, 7.6×10⁻⁷ M,7.7×10⁻⁷ M, 7.8×10⁻⁷ M, 7.9×10⁻⁷ M, 8×10⁻⁷ M, 8.1×10⁻⁷ M, 8.2×10⁻⁷ M,8.3×10⁻⁷ M, 8.4×10⁻⁷ M, 8.5×10⁻⁷ M, 8.6×10⁻⁷ M, 8.7×10⁻⁷ M, 8.8×10⁻⁷ M,8.9×10⁻⁷ M, 9×10⁻⁷ M, 9.1×10⁻⁷ M, 9.2×10⁻⁷ M, 9.3×10⁻⁷ M, 9.4×10⁻⁷ M,9.5×10⁻⁷ M, 9.6×10⁻⁷ M, 9.7×10⁻⁷ M, 9.8×10⁻⁷ M, 9.9×10⁻⁷ M, or 1×10⁻⁶ M)and/or to the the Fc portion of an Ig molecule with an affinity of 10⁻⁸M to 10⁻⁷ M (i.e. with an affinity of 1×10⁻⁸ M, 1.1×10⁻⁸ M, 1.2×10⁻⁸ M,1.3×10⁻⁸ M, 1.4×10⁻⁸ M, 1.5×10⁻⁸ M, 1.6×10⁻⁸ M, 1.7×10⁻⁸ M, 1.8×10⁻⁸ M,1.9×10⁻⁸ M, 2×10⁻⁸ M, 2.1×10⁻⁸ M, 2.2×10⁻⁸ M, 2.3×10⁻⁸ M, 2.4×10⁻⁸ M,2.5×10⁻⁸ M, 2.6×10⁻⁸ M, 2.7×10⁻⁸ M, 2.8×10⁻⁸ M, 2.9×10⁻⁸ M, 3×10⁻⁸ M,3.1×10⁻⁸ M, 3.2×10⁻⁸ M, 3.3×10⁻⁸ M, 3.4×10⁻⁸ M, 3.5×10⁻⁸ M, 3.6×10⁻⁸ M,3.7×10⁻⁸ M, 3.8×10⁻⁸ M, 3.9×10⁻⁸ M, 4×10⁻⁸M, 4.1×10⁻⁸ M, 4.2×10⁻⁸ M,4.3×10⁻⁸M, 4.4×10⁻⁸ M, 4.5×10⁻⁸M, 4.6×10⁻⁸ M, 4.7×10⁻⁸ M, 4.8×10⁻⁸ M,4.9×10⁻⁸ M, 5×10⁻⁸ M, 5.1×10⁻⁸ M, 5.2×10⁻⁸ M, 5.3×10⁻⁸ M, 5.4×10⁻⁸ M,5.5×10⁻⁸ M, 5.6×10⁻⁸ M, 5.7×10⁻⁸ M, 5.8×10⁻⁸ M, 5.9×10⁻⁸ M, 6×10⁻⁸ M,6.1×10⁻⁸ M, 6.2×10⁻⁸ M, 6.3×10⁻⁸ M, 6.4×10⁻⁸ M, 6.5×10⁻⁸ M, 6.6×10⁻⁸M,6.7×10⁻⁸ M, 6.8×10⁻⁸ M, 6.9×10⁻⁸ M, 7×10⁻⁸ M, 7.1×10⁻⁸ M, 7.2×10⁻⁸M,7.3×10⁻⁸ M, 7.4×10⁻⁸M, 7.5×10⁻⁸ M, 7.6×10⁻⁸ M, 7.7×10⁻⁸ M, 7.8×10⁻⁸ M,7.9×10⁻⁸ M, 8×10⁻⁸ M, 8.1×10⁻⁸ M, 8.2×10⁻⁸ M, 8.3×10⁻⁸ M, 8.4×10⁻⁸ M,8.5×10⁻⁸ M, 8.6×10⁻⁸ M, 8.7×10⁻⁸ M, 8.8×10⁻⁸ M, 8.9×10⁻⁸ M, 9×10⁻⁸ M,9.1×10⁻⁸ M, 9.2×10⁻⁸ M, 9.3×10⁻⁸ M, 9.4×10⁻⁸ M, 9.5×10⁻⁸M, 9.6×10⁻⁸ M,9.7×10⁻⁸ M, 9.8×10⁻⁸M, 9.9×10⁻⁸M, or 1×10⁻⁷ M).

In preferred embodiments the Ig binding moiety specifically binds to thesurface-exposed region of the C_(H)1 domain of an Ig molecule. The term“surface exposed region of an Ig molecule” preferably refers to thoseamino acids of an Ig molecule, which are free to specifically interactwith a binding moiety, if the binding moiety and the Ig molecule are insolution, preferably in a physiological solution. Preferably the“surface exposed regions” of an Ig molecule are those, which can elictan immune response, preferably a B cell specific immune response.Preferred is the surface-exposed region of the C_(H)1 domain of an IgGmolecule. Preferably, the Ig binding moiety interacts with the seventhβ-stand of the C_(H)1 domain of the Ig molecule. It is particularlypreferred that the Ig binding moiety specifically binds to an epitopeformed by amino acid positions 122-127 and/or 207-214 of an Ig moleculeaccording to EU index as in Kabat (see FIG. 12). Preferably, the Igbinding moiety specifically binds to an epitope formed by amino acidpositions 122-127 and/or 207-214 according to EU index as in Kabat of anhuman Ig γ1 according to SEQ ID NO: 4, human Ig γ2 according to SEQ IDNO: 5, human Ig γ3 according to SEQ ID NO: 6, human Ig γ4 according toSEQ ID NO: 7, mouse Ig γ1 according to SEQ ID NO: 8, mouse Ig γ2aaccording to SEQ ID NO: 9, mouse Ig γ2b according to SEQ ID NO: 10,mouse Ig γ3 according to SEQ ID NO: 11, and/or rat γ1 according to SEQID NO: 12, or variants thereof. Preferably, the Ig binding moiety has anaffinity to the C_(H)1 domain of an Ig molecule, more preferably to thesurface-exposed region of the C_(H)1 domain of an IgG molecule of below10⁻⁶ to below 10⁻⁹ M or the preferred affinities set out above in moredetail.

In the context of the present invention, the Ig binding moietypreferably comprises an immunoglobulin binding domain (IgBD).Preferably, the IgBD is derived from an Ig binding protein ofgram-positive bacteria, more preferably the IgBD is astreptococcus-derived IgBD. In preferred embodiments, the Ig bindingmoiety comprises a C_(H)1 binding-IgBD, preferably of a streptococcalstrain, more preferably a C_(H)1 binding-IgBD of streptococcal proteinG. It is further preferred that the Ig binding moiety comprises the C3IgBD of streptococcal protein G (the abbreviations “SpG-C3” or“SpG_(C3)”, are used interchangeably herein), more preferably comprisingan amino acid sequence according to SEQ ID NO: 1 or variants thereof. Inpreferred embodiments variants comprise an amino acid sequence of atleast 70% identity to the amino acid sequence of SpG-C3, preferably ofSEQ ID NO: 1, i.e. comprise an amino acid sequence of at least 70%, ofat least 71%, of at least 72% of at least 73% of at least 74%, of atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, or at least 98% to the amino acidsequence according to SEQ ID NO: 1. In particularly preferredembodiments, variants comprise an amino acid sequence of at least 94%identity to the amino acid sequence of SpG-C3, preferably of SEQ ID NO:1, i.e. comprise an amino acid sequence of at least 94%, at least 95%,at least 96%, at least 97%, or at least 98% to the amino acid sequenceaccording to SEQ ID NO: 1. Thus, it is preferred that variants ofSpG-C3, preferably of SEQ ID NO: 1, have between 1 and 14 amino acidssubstitutions, deletions and/or insertions, e.g. 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 substitutions, deletions and/or insertions.

It is preferred that the plasma half-life, preferably the initial and/orthe terminal plasma half-life, or the serum half-life, preferably theinitial and/or the terminal serum half-life, of the variant is notaltered with respect to the naturally occurring C_(H)1-binding IgBD,preferably the Ig binding protein of a gram-positive bacteria, on whichthe variant is based. In further preferred embodiments, the plasmahalf-life, preferably the initial and/or the terminal plasma half-life;or the serum half-life, preferably the initial and/or the terminal serumhalf-life, of the var the active compound iant is increased with respectto the naturally occurring C_(H)1-binding IgBD on which the variant isbased. Preferably, the plasma half-life, more preferably the initialand/or the terminal plasma half-life; or serum half-life, morepreferably the initial and/or the terminal serum half-life, is increasedby at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, more preferably by at least 80%.

It is also preferred that the bioavailability of the variant is notaltered with respect to the naturally occurring C_(H)1-binding IgBD. Itis further preferred that the bioavailability of the variant isincreased with respect to the naturally occurring C_(H)1-binding IgBD.Preferably, the bioavailability of the variant is increased by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, more preferably by at least 80%.

In preferred embodiments such variant comprises amino acid exchanges,insertions, deletions, or N- or C-terminal truncations, or anycombination of these changes, which may occur at one or several sites.Optionally such variants may alter the binding properties of the IgBD,preferably by increasing the binding affinity of the IgBD to the Igmolecule. An increased binding affinity to an Ig molecule may beachieved by preventing the binding of the IgBD to the Fc- or theFab-fragment (“C3-Fc” or “C3-Fab”, respectively), e.g. by exchanging oneor more amino acid positions to contain the amino acid alanine.Preferably, the Fab-binding of the C3 IgBD of streptococcal protein G isprohibited by the amino acid exchanges Thr10Ala, Lys12Ala, and Glu14Ala,as shown in SEQ ID NO: 2 (C3-Fc). The Fc-binding of the C3 IgBD ofstreptococcal protein G is prohibited by the amino acid exchangesGlu26Ala, Lys27Ala, and Lys30Ala, as shown in SEQ ID NO: 3 (C3-Fab).

In embodiments of the first aspect of the present invention, thepharmaceutically active moiety comprises a biological and/or chemicalpharmaceutical. Preferably, the pharmaceutically active moiety comprisesa biological such as but not limited to pharmaceutically, preferablytherapeutically, active peptides, polypeptides, or proteins produced viabiotechnological means. Suitable biologicals include but are not limitedto hormones (e.g. insulin, hGH, FSH, Glucagon-like peptide 1,parathyroid hormone, calcitonin, lutropin, glucagon), blood factors(e.g. factor VIII, factor IX, factor XI), growth factors (e.g.erythropoietin, G-CSF/GM-CSF, IGF-1), interferons (e.g. IFN-α, IFN-β,IFN-γ), interleukins (e.g. IL-2, IL-11, IL-1Ra), coagulation factors(e.g. factor VIII, factor IX, factor VIIa, thrombin), thrombolytics andanti-coagulants (e.g. t-PA, hirudin, activated protein C), enzymes (e.g.α-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase,urate oxidase, DNase), vaccines (e.g. parasitic, fungal, bacterial, orviral antigens such as e.g. hepatitis B surface antigens),antigen-binding molecule such as antibodies and antibody fragments (e.g.IgG, Fab), and fusion proteins thereof (e.g. TNFR2-Fc, TMP-Fc,CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

In preferred embodiments, the pharmaceutically active moiety does notcomprise the N-terminal domain of the diphteria toxin, acellulose-binding domain (CBD), diagnostic proteins, in particular afirefly luciferase and/or the green fluorescent protein (GFP) ofAequorea victoria. Diagnostic proteins are those, which are capable tofluoresce.

In the context of the present invention it is particularly preferredthat the pharmaceutically active moiety comprises an antigen-bindingmolecule such as an immunoglobulin molecule. Preferably, theantigen-binding molecule is selected from the group consisting of anantibody fragment, a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment,a heavy chain antibody, a single-domain antibody (sdAb), a single-chainvariable fragment (scFv), a di-scFv, a bispecific T-cell engager(BITEs), a diabody, a single-chain diabody, a DART molecule, a triplebody, an alternative scaffold protein, and a fusion proteins thereof. Inparticularly preferred embodiments the antigen-binding molecule is ascFv or a diabody or a fusion protein comprising a scFv or a diabody.Preferably, the antigen-binding molecule does not comprise acellulose-binding domain (CBD).

Additionally or alternatively, the antigen-binding molecule may furthercomprise a radioactive moiety, a cytotoxic drug, a chelating moiety, aphotosensitizer, or an imaging reagent.

In preferred embodiments the antigen-binding molecule comprises aradioactive moiety, i.e. a radionuclide. The radioactive moiety may bean isotpe of F, Br, Mn, Co, Ga, As, Zr, P, C, S, H, I, In, Lu, Cu, Rh,Bi, At, Y, Re, Ac, Tc, or Hg atom. The radioactive moiety labels theantigen-binding molecule radioactively allowing for its detection, e.gin the human body, rendering it not only useful for diagnosticapproaches (radioimmunodetection: RAID) but also suitable in therapeuticapplications (radioimmunotherapy: RAIT).

Photosensitizers are chemical compounds capable of light emission orformation of free radicals and singlet oxigen after being excited bylight of a specific wavelength. Photosensitizer are used e.g. forphotodynamic therapy. In preferred embodiments photosenitizer includebut are not limited to compounds of the porphyrin family, texaphyrinfamily, the chlorin family and the phthalocyanine family, in particularincluding HpD, ALA, M-ALA, Vertiporfin, Lutexaphyrin, Temoporfin,Talaporfin, HPPH, Phthalocyanine, and Napthalocyanine.

Imaging reagents include bioluminescent, chemiluminescent andfluorescent imaging reagent such as but not limited to luciferase fromRenilla reniformis and/or Metridia Longa, peroxalate, polymethines (e.g.cyanine dyes such as Cy3, Cy5, Cy5.5, Cy7) squaraine derivatives,phthalocyanine, porphhyrin derivatives, and BODIPY analogous (BODIPY FL,BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY 581/591, BODIPY 630/650,BODIPY 650/665), as well as fluorescent proteins such as but not limitedto CFP, BFP, YFP, DsRED (Chudakov et al. (2010) Physiol. Rev.90:1103-1163). Preferably, the fluorescent protein is not GFP.

In preferred embodiments the antigen-binding molecule is a cytotoxicdrug which has a toxic effect on cells such as but not limited toantimitotic drugs, drugs prohibiting cell growths and drugs causing celldeath. Non-limiting examples of cytotoxic drugs are alkylating agents(e.g. cisplatin, carboplatin, oxaloplatin, mechlorethamine,cyclophosphamide, chlorambucil), anti-metabolites (5-fluorouracil(5-FU), capecitabine (Xeloda®), 6-mercaptopurine (6-MP), methotrexate,gemcitabine), plant alkaloids (e.g. ajmaline, atropine, scopolamine,hyoscyamine, vinca alkaloids, codeine cocaine colchicine morphine,reserpine, tubocurarine, physostigmine, quinidine, quinine, emetine,ergot alkaloids), antitumor antibiotics (e.g. actinomycin-D, bleomycin,and mitomycin-C, mitoxantrone, and anthracyclines such as daunorubicin,doxorubicin), topoisomerase inhibitors (e.g. topotecan,irinotecanetoposide (VP-16) and teniposide), and mitotic inhibitors(estramustine, taxanes such as paclitaxel and docetaxel, epothilonessuch as ixabepilone, and vinca alkaloids such as vinblastine,vincristine, vindesine and vinorelbine).

The antigen-binding molecule may further comprise a chelating moietycapable of binding at least one metal ion, such as but not limited tocalcium, magnesium, iron, aluminium, zinc, copper, arsenic, lead,thallium, and mercury ions, by chelation. Such chelating moiety maycomprise ethylenediamine tetraacetic acid (EDTA), ethylenediaminetetraacetic acid (calcium disodium versante) (CaNa₂-EDTA), dimercaprol(BAL), dimercaptosuccinic acid (DMSA), dimercapto-propane sulfonate(DMPS), ferritin, deferoxamine and deferasirox, deferiprone(1,2-dimethyl-3-hydroxyl-4-pyridinone), DOTA, DTPA, DADT, DADS, DO3A,N2S2MAMA, Triamidethiol, phosphonates, organic gadolinium complexes,penicillamine, and antibiotic drugs of the tetracycline family. Achelating moiety is of particular interest in chelating therapy, e.g. inthe treatment of atherosclerosis, rheumatoid arthritis, and poisoningsuch as mercury poisoning, copper toxicity, gold toxicity, arsenicpoisoning, lead poisoning, acute iron poisoning, and iron overload.Chelating moieties are also important for radiotherapy.

Preferably, the antigen binding molecule is a fusion protein, whichadditionally or alternatively further comprises a proapoptotic protein,an immuno-(co)stimulatory protein, immuno-suppressive protein, acytokine (e.g. interleukins and/or interferons), a chemokine (e.g. anα-, β-, or γ-chemokine), a toxin, a growth factor or an enzyme,preferably a RNase, a prodrug-converting enzmye or a kinase (e.g. AGCkinases, CaM kinases, CK1 kinases, CMGC kinases, STE kinases, TKkinases, and TKL kinases).

In preferred embodiments proapoptotic protein include but are notlimited to Bid, Bik, Puma, and Bim, and proapoptic cytokines (deathligands) such as but not limited to TNF, TRAIL, and FasL.

In preferred embodiments immuno-(co)stimulatory protein include but arenot limited to B7.1, B7.2, 4-1BBL, LIGHT, ICOSL, GITR, CD40, OX40L, andCD70.

Immuno-suppressive proteins preferably include but are not limited toIL1-Ra and toxins preferably include but are not limited to Pseudomonasexotoxin A and ricin. Preferably, the toxin is not diphteria toxin.

In preferred embodiments, cytokines are interleukins and/or interferons.Interleukins (IL) include but are not limited to Interleukin-1,Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5,Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9,Interleukin-10, Interleukin-11, Interleukin 12, Interleukin-13,Interleukin-14, Interleukin-15, Interleukin-16, Interleukin-17,Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21,Interleukin-22, Interleukin-23, Interleukin-24, Interleukin-25,Interleukin-26 Interleukin-27, Interleukin-28, Interleukin-29,Interleukin-30, Interleukin-31, Interleukin-32, Interleukin-33,Interleukin-34 and Interleukin-35. Interferons (IFN) include but are notlimited to interferon type I (e.g. IFN-α, IFN-β and IFN-ω), interferontype II (e.g. IFN-γ), and interferon type III. In particular includedare interferon A1, interferon A2, interferon A4, interferon A5,interferon A6, interferon A7, interferon A8, interferon A10, interferonA13, interferon A14, interferon A16, interferon A17, interferon A21,interferon B1, TNF, TRAIL, and FasL.

In preferred embodiments growth factors include but are not limited toAdrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bonemorphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF),Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growthfactor (FGF), Glial cell line-derived neurotrophic factor (GDNF),Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophagecolony-stimulating factor (GM-CSF), Growth differentiation factor-9(GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor(HDGF), Insulin-like growth factor (IGF), Migration-stimulating factorMyostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins,Platelet-derived growth factor (PDGF), Thrombopoietin (TPO),Transforming growth factor alpha (TGF-α), Transforming growth factorbeta (TGF-β), Vascular endothelial growth factor (VEGF), Wnt SignalingPathway, and placental growth factor (PlGF).

RNAses include endoribonucleases such as but are not limited to RNase A,RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1,RNase T2, RNase U2, RNase V1, and RNase V, and exoribonucleases such asbut not limited to Polynucleotide Phosphorylase (PNPase), RNase PH,RNase II, RNase R, RNase D, RNase T, Oligoribonuclease ExoribonucleaseI, and Exoribonuclease II.

Pro-drug-converting enzymes include but are not limited to esterasessuch as but not limited to acetylesterase, thiolester hydrolases,phosphoric monoester hydrolases, phosphoric diester hydrolases,triphosphoric monoester hydrolases, sulfuric ester hydrolases(sulfatases), diphosphoric monoester hydrolases, and phosphoric triesterhydrolases; phosphatases such as but not limited to tyrosine-specificphosphatases, serine/threonine specific phosphatases, dual specificityphosphatases, histidine phosphatase, and lipid phosphatase; andreductases such as but not limited to 5-alpha reductase, dihydrofolatereductase, HMG-CoA reductase, methemoglobin reductase, ribonucleotidereductase, thioredoxin reductase, E. coli nitroreductase,methylenetetrahydrofolate reductase, and carboxypeptidase G2, cytosinedeaminase, nitroreductase, thymidine kinase.

Kinases include but are not limited to AGC kinases such as PKA, PKC andPKG, CaM kinases such as calcium/calmodulin-dependent protein kinasesand serine/threonine protein kinases (e.g. DAPK2), CK1 such as thecasein kinase 1 group, CMGC such as CDK, MAPK, GSK3 and CLK kinases, STEsuch as homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases,tyrosine kinases (TK), the tyrosine-kinase like group of kinases (TKL),receptor-associated tyrosine kinases, MAP kinases, and histidinekinases.

In particularly preferred embodiments the pharmaceutically active moietyis a peptide-linked or a disulfide-linked single-chain diabody. It isparticularly preferred that the pharmaceutically active moiety is asingle-chain diabody with a first specificity (A) directed against atarget molecule, and a second specificity (B) directed against aneffector molecule. Preferably, the single-chain diabody comprises thestructure [VH(A)-VL(B)-P-VH(B)-VL(A)] or [VL(B)-VH(A)-P-VL(A)-VH(B)]. Inpreferred embodiments the first specificity (A) is directed against atumor-associated antigen or an antigen of a pathogen. Preferably, thetumor-associated antigen is selected from the group consisting of CEA,EGFR, HER2, HER3, HER4, VEGFRs, integrin receptor family, fibroblastactivation protein, galectin, EpCAM, CEA, CD44, CD44v, CD2, CD5, CD7,CD19, CD20, CD21, CD22, CD24, CD25, CD30, CD33, CD38, CD40, CD52, CD56,CD71, CD72, CD73, CD105, CD117, CD123, c-Met, PDGFR, IGF1-R, HMW-MAA,TAG-72, GD2, GD3, GM2, folate receptor, Leg, MUC-1, MUC-2, PSMA, PSCAand uPAR. In further preferred embodiments the second specificity (B) isdirected against molecules of cell membranes, cytokines, chemokines,growth factors, proteins of the complement system, proteins of thecoagulation system, fibrinolytic proteins, enzymes which are able toconvert the inactive precursor of a drug into an active drug on thetarget structure, peptide hormones, steroid hormones, the constant partof an immunoglobulin, cytotoxic peptide, and pharmaceuticals.Preferably, the second specificity (B) is directed against molecules onthe cell membrane of lymphocytes, macrophages, monocytes orgranulocytes, more preferably against molecules on the cell membrane ofT-cells. It is particularly preferred that the second specificity (B) isdirected against CD3, more preferably against the extracellular regionof CD3.

In further embodiments of the present invention the Ig binding moietyand the pharmaceutically active moiety are connected via covalent ornon-covalent bond(s). It is particularly preferred that the Ig bindingmoiety and the pharmaceutically active moiety are connected directly orindirectly via one or more linkers. Preferably, the one or more linkerscomprise peptide linkers, more preferably flexible peptide linkers. Inpreferred embodiments, a peptide linker according to the presentinvention has a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 amino acids, preferably of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 amino acids. Preferably, a peptide linkeraccording to the present invention has a maximum length of at least 100,95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 aminoacids or less. Preferably, the linker has a length of 1-40, preferablyof 5-20, more preferably of 18-12, most preferably of 10 amino acids. Inpreferred embodiments of the present invention, the peptide linker hasan increased content of small amino acids, in particular of glycinsand/or alanines, and/or hydrophilic amino acids such as serines,threonines, asparagines and glutamines. Preferably, more than 20%, 30%,40%, 50%, 60% or more of the amino acids of the peptide linker are smalland/or hydrophilic amino acids. Preferably the amino acids of the linkerare selected from glycines and serines. In further preferredembodiments, the peptide linker of the present invention isnon-immunogenic; in particularly preferred embodiments, the peptidelinker is non-immunogenic to humans. A peptide linker having thesequence GGSGGGGSGG is particularly preferred.

In preferred embodiments the Ig binding moiety comprises a streptococcalIgBD, more preferably the C3 IgBD of streptococcal Protein G (SpG-C3),which is connected via a flexible linker to the pharmaceutically activemoiety, preferably to an antigen binding molecule selected from thegroup consisting of an antibody fragment, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, a heavy chain antibody, a single-domainantibody (sdAb), a single-chain variable fragment (scFv), a di-scFv, abispecific T-cell engager (BITEs), a diabody, a single-chain diabody, aDART molecule, a triple body, an alternative scaffold protein, and afusion protein thereof.

In particularly preferred embodiments of the first aspect of the presentinvention, the complex of the present invention comprises an amino acidsequence according to SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 orvariants thereof. Preferably, such variant has a sequence identity of atleast 94%, i.e. of at least 94%, at least, 95%, at least 96%, at leat97%, at least 98%, at least 99% sequence identity, to an amino acidsequence according to SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

In further embodiments the one or more peptide linkers comprise one ormore cleavage sites, preferably one or more endopeptidase cleavagesites. It is preferred that the cleavage site allows for the release ofthe pharmaceutically active moiety once the intended destination isreached. Preferably, an endopeptidase cleavage site relates to cleavagecite within the amino acid sequence where this sequence is cleaved or iscleavable by an endopeptidase such as but not limited to trypsin,pepsin, elastase, thrombin, collagenase, furin, thermolysin,endopeptidase V8, metalloproteinases and cathepsins.

In a second aspect, the present invention provides a nucleic acidmolecule comprising a sequence encoding the complex of the first aspect.Preferably such nucleic acid molecule comprises a DNA and/or RNAmolecule.

In a third aspect, the present invention provides a vector comprisingthe nucleic acid of the second aspect. It is understood that suitablevectors include but are not limited to plasmids, cosmids, phages,viruses and/or artificial chromosomes.

In a fourth aspect, the present invention provides an isolated cellcontaining the complex of the first aspect and/or the nucleic acidmolecule of the second aspect and/or the vector of the third aspect. Itis understood that such cell includes but is not limited to prokaryotic(e.g. a bacterial cell) or eukaryotic cells (e.g. a fungal, plant oranimal cell).

In a fifth aspect, the present invention provides a compositioncomprising the complex of the first aspect, the nucleic acid of thesecond aspect, the vector of the third aspect and/or the cell of thefourth aspect and a pharmaceutical acceptable carrier and/or excipient.Preferably, such composition is a pharmaceutical composition. Inpreferred embodiments the pharmaceutical composition further comprises apharmaceutically acceptable carrier and/or excipient and optionally oneor more additional active substances. Preferably, the composition of thefifth aspect contains a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier and/or excipient so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

The pharmaceutical compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The pharmaceuticalcomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

For preparing pharmaceutical compositions of the present invention,pharmaceutically acceptable carriers can be either solid or liquid.Solid form compositions include powders, tablets, pills, capsules,lozenges, cachets, suppositories, and dispersible granules. A solidexcipient can be one or more substances, which may also act as diluents,flavouring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material. In powders, the excipient is preferably afinely divided solid, which is in a mixture with the finely dividedinhibitor of the present invention. In tablets, the active ingredient ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.Suitable excipients are magnesium carbonate, magnesium stearate, talc,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. For preparing suppositories, a low melting wax,such as a mixture of fatty acid glycerides or cocoa butter, is firstmelted and the active component is dispersed homogeneously therein, asby stirring. The molten homogeneous mixture is then poured intoconvenient sized moulds, allowed to cool, and thereby to solidify.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

Liquid form compositions include solutions, suspensions, and emulsions,for example, water, saline solutions, aqueous dextrose, glycerolsolutions or water/propylene glycol solutions. For parenteral injections(e.g. intravenous, intraarterial, intraosseous infusion, intramuscular,subcutaneous, intraperitoneal, intradermal, and intrathecal injections),liquid preparations can be formulated in solution in, e.g. aqueouspolyethylene glycol solution. A saline solution is a preferred carrierwhen the pharmaceutical composition is administered intravenously.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form the composition may be subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged composition, the package containing discrete quantities ofthe composition, such as packaged tablets, capsules, and powders invials or ampoules. Also, the unit dosage form can be a capsule, aninjection vial, a tablet, a cachet, or a lozenge itself, or it can bethe appropriate number of any of these in packaged form.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents.

Furthermore, such pharmaceutical composition may also comprise otherpharmacologically active substance such as but not limited to adjuvantsand/or additional active ingredients. Adjuvants in the context of thepresent invention include but are not limited to inorganic adjuvants,organic adjuvants, oil-based adjuvants, cytokines, particulateadjuvants, virosomes, bacterial adjuvants, synthetic adjuvants, orsynthetic polynucleotides adjuvants.

In a sixth aspect, the present invention provides the complex of thefirst aspect of the present invention as described in detail above, forthe use in extending the serum half-life and/or the plasma half-life. Itis preferred that the complex of the first aspect of the presentinvention is for the use in extending the initial and/or terminal serumhalf-life. In preferred embodiments the serum half-life, more preferablythe initial and/or terminal serum half-life, of the pharmaceuticallyactive moiety is prolonged. Preferably, the serum half-life, morepreferably the initial and/or terminal serum half-life, of thepharmaceutically active moiety is prolonged due to its complexing to animmunoglobulin-binding moiety, preferably to an IgBD, more preferably tothe C3-IgBD of streptococcal Protein G.

It is further preferred that the complex of the first aspect of thepresent invention is for the use in extending the initial and/orterminal plasma half-life. In preferred embodiments the plasmahalf-life, more preferably the initial and/or terminal plasma half-life,of the pharmaceutically active moiety is prolonged. Preferably, theplasma half-life, more preferably the initial and/or terminal plasmahalf-life, of the pharmaceutically active moiety is prolonged due to itscomplexing to an immunoglobulin-binding moiety, preferably to an IgBD,more preferably to the C3-IgBD of streptococcal Protein G.

In a seventh aspect, the present invention provides the complex of thefirst aspect of the present invention as described in detail above foruse as a medicament. In preferred embodiments the complex is for use inmedicine, i.e. for use in the prophylaxis, treatment or diagnosis of adisorder or disease such as but not limited to autoimmune diseases,allergic diseases, cancer type diseases, cutaneous conditions, endocrinediseases, eye diseases and disorders, genetic disorders, infectiousdiseases, intestinal diseases, neurological disorders, and mentalillness. Exemplified, autoimmune diseases include but are not limited toDiabetes mellitus type 1, rheumatoid arthritis, psoriasis, CrohnsDisease, autoimmune cardiomyopathy, autoimmune hepatitis, Hashimoto'sthyroiditis, and Sjogern's syndrome. Exemplified, allergic diseasesinclude but are not limited to allergic rhinitis, asthma, atopic eczema,anaphylaxis, insect venom allergies, drug allergies, and food allergies.Exemplified, cancer type diseases include but are not limited to Basalcell carcinoma, Bladder cancer, Bone cancer, Brain tumor, Breast cancer,Burkitt lymphoma, Cervical cancer, Colon Cancer, Cutaneous T-celllymphoma, Esophageal cancer, Retinoblastoma, Gastric (Stomach) cancer,Gastrointestinal stromal tumor, Glioma, Hodgkin lymphoma, Kaposisarcoma, Leukemias, Lymphomas, Melanoma, Oropharyngeal cancer, Ovariancancer, Pancreatic cancer, Pleuropulmonary blastoma, Prostate cancer,Throat cancer, Thyroid cancer, and Urethral cancer. Exemplified,cutaneous conditions include but are not limited to Acne, Dermatitis,Eczema, conditions of the skin appendages, conditions of thesubcutaneous fat, disturbances of pigmentation, epidermal nevi,epidermal neoplasms, epidermal cysts, erythemas, frostbitesgenodermatoses, mucinoses, neurocutaneous conditions (e.g.Wiskott-Aldrich syndrome), and psoriasis. Exemplified, endocrinediseases include but are not limited to Diabetes mellitus type 1 andtype 2, Osteoporosis, and Cushing's disease. Exemplified, geneticdisorders include but are not limited to color blindness, cysticfibrosis, Down syndrome, Sickle-cell disease, and Turner syndrome.Exemplified, infectious diseases include but are not limited toinfections diseases caused by viruses, bacteria, worms, prions or otherpathogens or parasites such as African sleeping sickness, AIDS, HIVinfection, Anthrax, Borreliosis, Calicivirus infection (Norovirus andSapovirus), Chickenpox, Chlamydia infection, Cholera, Clostridiuminfection, Colorado tick fever (CTF), common cold, Creutzfeldt-Jakobdisease, Dengue fever (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola,Enterovirus infection, infections with Human herpesvirus 6 (HHV-6) andHuman herpesvirus 7 (HHV-7), Gonorrhea, Streptoccocal infections (groupA and B), Hand, foot and mouth disease (HFMD), Helicobacter pyloriinfection, Hepatitis (A, B, C, and D), Herpes infection, Papillomavirusinfection, Parainfluenza virus infection, Influenza, Lassa fever,Marburg fever, Measles, Meningitis, Mumps, Pasteurellosis, Pediculusinfection, Plague, Pneumococcal infection, Respiratory syncytial virusinfection, Rotavirus infection, Rubella virus infection, Salmonella foodpoisoning and infection, SARS, Scabies infections, Schistosomiasis,Smallpox, Staphylococcal food poisoning and infection, Syphilis,Tetanus, Trichophyton infection, Tuberculosis, Typhus, Venezuelan equineencephalitis, and Yellow fever. Exemplified, intestinal diseases includebut are not limited to Gastroenteritis, Ileus, Ileitis, Colitis,Appendicitis, Coeliac disease, Irritable bowel syndrome, Diverticulardisease, Diarrhea, Polyp, and Ulcerative colitis. Exemplified,neurological disorders include but are not limited to AmyotrophicLateral Sclerosis (ALS), Alzheimer's disease, Brain damage,Creutzfeldt-Jakob disease, Cushing's syndrome, Dyslexia, Encephalitis,Epilepsy, Headache, Huntington's disease, Migraine, Multiple sclerosis,Parkinson's disease, Polio, Rabies, Schizophrenia, and Stroke.Exemplified, mental illness include but are not limited to Acute stressdisorder, attention-deficit hyperactivity disorder (ADHD), Autisticdisorder, Borderline personality disorder, Bulimia nervosa, Burn Out,Schizophrenia, Depression, Cognitive disorder, Communication disorder,Eating disorder, Kleptomania, Learning disorders, Male erectiledisorder, Melancholia, Obsessive-compulsive disorder (OCD), ParanoiaPathological gambling, Posttraumatic stress disorder (PTSD), Psychoticdisorder, Hypersomnia, Insomnia, and Tourette's syndrome.

The following examples are merely illustrative of the present inventionand should not be construed to limit the scope of the invention asindicated by the appended claims in any way.

EXAMPLES Example 1 Construction and Production of scDb-IgBD FusionProteins

DNA encoding the IgBDs (IgBD SpA_(B), SpA_(D), SPA_(EZ4), SPG_(C3), andPpL_(C4*)) including a hexahistidyl-tag at the C-terminus weresynthesized by GeneArt (Regensburg, Germany) adding a NotI at the 5′ endand an EcoRI and XbaI site at the 3′ end. IgBD SpA_(B) was cloned intomammalian expression vector pSecTagAHis scDb-CEACD3-ABD-L (Hopp et al.(2010) Protein Eng. Des. Sel. 23:827-834) cut with NotI and XbaI. TheIgBD SpA_(D), SpA_(EZ4), SpG_(C3), and PpL_(C4*) were then cloned intoscDb-CEACD3-SpA_(B) as NotI-EcoRI fragments substituting the SpA_(B)IgBD. Composition of the scDb-IgBD fusion proteins are given in FIG. 2a. HEK293 cells were stably transfected and the fusion proteinsscDb-SpA_(B), scDb-SpA_(D), scDb-SpA_(EZ4), scDb-SpG_(C3), andscDb-PpLC_(4*) were purified from cell culture supernatant by IMACessentially as described previously (Müller et al. (2007) J. Biol. Chem.282:12650-12660). Yields of 2 to 22 mg/L supernatant were obtained.SDS-PAGE of purified fusion proteins was performed. Two microgramsproteins were analyzed per lane and the gel was stained with Coomassiebrilliant blue G-250 (M, molecular weight standards). SDS-PAGE analysisrevealed a single band under reducing and non-reducing conditions (FIG.2 b). Compared with unmodified scDb, the molecular mass was increased byapproximately 5 kDa under reducing conditions.

Example 2 Construction and Production of scFv-IgBD Fusion Proteins

DNA encoding the IgBDs (IgBD SpA_(B), SpA_(D), SPA_(EZ4), SPG_(C3), andPpL_(C4*)) including a hexahistidyl-tag at the C-terminus weresynthesized by GeneArt (Regensburg, Germany) adding a NotI at the 5′ endand an EcoRI and XbaI site at the 3′ end. The DNA was digested with NotIand EcoRI and cloned into vector pSecTagA-scFvCEA-4-1BBL (Müller et al.(2008) J. Immunol. 31:714-722). Composition of the scFv-IgBD fusionproteins are given in FIG. 2 a. HEK293 cells were stably transfected andthe fusion proteins scFv-SpA_(B), scFv-SpA_(D), scFv-SpA_(EZ4),scFv-SpG_(C3), and scFv-PpL_(C4*) were purified from cell culturesupernatant by IMAC essentially as described previously (Müller et al.,(2007) J. Biol. Chem. 282:12650-12660). SDS-PAGE of purified fusionproteins was performed. Two micrograms proteins were analyzed per laneand the gel was stained with Coomassie brilliant blue G-250 (M,molecular weight standards). SDS-PAGE analysis revealed a single bandunder reducing and non-reducing conditions (FIG. 2 c). Compared withunmodified scFv, the molecular mass was increased by approximately 5 kDaunder reducing conditions.

Example 3 Size Exclusion Chromatography (SEC)

Purity and stokes radii of the scDb-IgBD and scFv-IgBD fusions proteinswere analyzed by HPLC size exclusion chromatography using a BioSuite 250(Waters Corporation, Milford, USA) and a flow rate of 0.5 ml/min (FIG. 2d-g). The following standard proteins were used: thyroglobulin,β-amylase, bovine serum albumin, carbonic anhydrase, cytochrome c. Allfusion proteins showed a single peak corresponding to monomericmolecules. The measured Stokes radii of the fusion proteins were in therange of 2.3 to 2.7 nm. Interestingly, the Stokes radii of the scFv-IgBDfusion proteins were similar to those of the scDb-IgBD fusion proteins,while the unmodified scFv had a Stokes radius of 1.2 nm (see also FIG.8).

Example 4 Binding of scDb-IgBD and scFv-IgBD Fusion Proteins to CEA inELISA

Increasing concentrations of the scDb-IgBD (a) or scFv-IgBD (b) fusionproteins were analyzed for binding to immobilized CEA by ELISA.Carcinoembryonic antigen (CEA) (300 ng/well) was coated overnight at 4°C. and remaining binding sites were blocked with 2% (w/v) dry milk/PBS.Purified recombinant scDb, scFV, as well as scDb-IgBD and scFv-IgBDfusion proteins were titrated in duplicates and incubated for 1 h at RT.Detection was performed with mouse HRP-conjugated anti-His-tag antibodyusing TMB substrate (0.1 mg/ml TMB, 100 mM sodium acetate buffer pH 6.0,0.006% H₂O₂). The reaction was stopped with 50 μl of 1 M H₂SO₄.Absorbance was measured at 450 nm in an ELISA-reader.

Example 5 Binding of scDb-IgBD Fusion Proteins to Human and Mouse IgG,Human Fab- and Fc-Fragments

The fusion proteins scDb-SpG_(C3), scDb-SpA_(B), scDb-SpA_(D),scDb-SpA_(EZ4), and scDb-PpL_(C4*) were analyzed for binding toimmobilized human serum IgG as well as Fab and Fc fragments thereof byELISA. Human or mouse IgG, human Fab or human Fc fragments (100 ng/well)was coated overnight at 4° C. and remaining binding sites were blockedwith 2% (w/v) dry milk/PBS. Purified recombinant antibodies and serumsamples were titrated in duplicates and incubated for 1 h at RT.Detection was performed with mouse HRP-conjugated anti-His-tag antibodyusing TMB substrate (0.1 mg/ml TMB, 100 mM sodium acetate buffer pH 6.0,0.006% H₂O₂). The reaction was stopped with 50 μl of 1 M H₂SO₄.Absorbance was measured at 450 nm in an ELISA-reader. Strongest bindingto human serum IgG (huIgG), as well as human Ig Fc (huIgFc) was observedfor scDb-SpG_(C3) (FIG. 4 b). Also, scDb-SpA_(B), scDb-SpA_(D),scDb-SpA_(EZ4) were able to bind to huIgG and huIgFc, however theirbinding was weaker than the binding of scDb-SpG_(C3). Hardly any bindingcould be observed for scDb-PpL_(C4*). Binding to huIgFab could beobserved for scDb-SpG_(C3), whilst all other fusion protein showedhardly any binding to huIgFab. The fusion proteins scDb-SpG_(C3),scDb-SpA_(B), scDb-SpA_(D), and scDb-SpA_(EZ4) were also able to bind tohuIgM (FIG. 4 b) with scDb-SpA_(B) exhibiting the strongest binding(FIG. 4 b). In addition, the fusion proteins scDb-SpA_(B), scDb-SpA_(D),and scDb-SpA_(EZ4) also showed binding to huIgA.

Binding of all fusion proteins (except scDb-PpL_(C4*)) was also seenwith mouse serum IgG (molgG) as well as mouse Ig Fc (molgFc), althoughbinding was generally weaker than that seen for the human IgGs (FIG. 4a). Binding to and mouse Ig Fab (molgFab) fragments was only observedfor scDb-SpG_(C3).

Example 6 Affinity Measurements

Affinities of scDb-IgBD fusion proteins for human and mouse serum IgGwell as Fab and Fc fragments at neutral or acidic pH were determined byquartz crystal microbalance measurements (Attana A-100 C-Fast system).IgGs as well as Fab and Fc fragments were chemically immobilized on anLNB (low nonspecific binding) carboxyl sensor chip according to themanufacturer's protocol at a density resulting in a signal increase of65-95 Hz. Binding experiments were performed in PBST (0.1% Tween 20) pH7.4 or pH 6.0 with at a flow rate of 25 μl/min. The chip was regeneratedwith 25 μl 10 mM glycine-HCl pH 3.0. Before each measurement, a baselinewas measured which was subtracted from the binding curve. Data werecollected by Attester 3.0 (Version 3.1.1.8, Attana, Stockholm, Sweden)and analyzed by Attache Office Evaluation Software (Version 3.3.4,Attana, Stockholm, Sweden), using a mass transport model for curvefitting (see FIG. the active compound 5, FIG. 5). Strong binding in thelow nanomolar range to human and mouse IgG as well as IgG-Fc wasobserved for the different SpA-IgBD and the SpG_(C3) fusion proteins.Binding to human and mouse Fab fragments was only observed forscDb-SpG_(C3). The binding of the scDb-SpA-IgBD fusion protein was foundto be is pH-dependent and was strongly reduced at pH 6. For example,lowering the pH from 7.4 to 6.0 resulted in an approximately 45-foldreduced affinity of scDb-SpA_(B) for human serum IgG and a 43-foldreduced affinity for mouse serum IgG. A pH-dependent binding may have adirect influence on FcRn-mediated recycling, which requires that the SpAfusion protein stays bound to IgG-FcRn complexes in the acidicenvironment of the early endosome (pH˜6.3 to 6.8) and tubular recyclingendosomes (pH˜6.5). In contrast, similar or even an increased bindingaffinity of scDb-SpG_(C3) to human IgFc, human IgFab, mouse IgG andmouse IgFab were observed by lowering the pH value from 7.4 to 6 (FIG.5).

Example 7 Pharmacokinetics

CD1 mice were purchased from Elevage Janvier (Le Genest St. Isle,France). Animal care and all experiments performed were in accordancewith federal guidelines and have been approved by university and stateauthorities. CD1 mice (8-16 weeks, weight between 30-40 g) received ani.v. injection of 25 μg a scDb-IGBD or a scFv-IGBD fusion protein in atotal volume of 150 μl. In time intervals of 3 min, 30 min, 1 h, 2 h, 6h, 1 day, and 3 days blood samples (50 μl) were taken from the tail andincubated on ice. Clotted blood was centrifuged at 13,000 g for 10 min,4° C. and serum samples stored at −20° C. The active compound Serumconcentrations of CEA-binding recombinant antibodies were determined byELISA. Carcinoembryonic antigen (CEA) (300 ng/well) or IgG (500 ng/well)was coated overnight at 4° C. and remaining binding sites were blockedwith 2% (w/v) dry milk/PBS. Purified recombinant antibodies and serumsamples were titrated in duplicates and incubated for 1 h at RT. Fordetermination of pH dependence of binding, all incubation and washingsteps were performed with PBS adjusted to the indicated pH. Detectionwas performed with mouse HRP-conjugated anti-His-tag antibody using TMBsubstrate (0.1 mg/ml TMB, 100 mM sodium acetate buffer pH 6.0, 0.006%H₂O₂). The reaction was stopped with 50 μl of 1 M H₂SO₄. Absorbance wasmeasured at 450 nm in an ELISA-reader. For comparison, the first value(3 min) was set to 100%. Half-life of scDb-IgBD and scFv-IgBD fusionproteins was analyzed after a single i.v. injection into CD1 mice. Theinitial plasma half-live (t_(1/2)α), the terminal plasma half-life(t_(1/2)β) and the bioavailability (AUC) were calculated for scDb-IgBDand scFv-IgBD fusion proteins using Excel (FIG. 8). For statistics,Student's t-test was applied. The bioavailability of all fusion proteinswas increased in comparison to the non-fused scDb or scFv, respectively.The highest increase in the bioavailability was obtained byscDb-SpG_(C3) and scFv-SpG_(C3) in comparison to the non-fused scDb orscFv, respectively, with scDb-SpG_(C3) exhibiting a 36-fold increase andscFv-SpG_(C3) exhibiting a 65-fold increase in their bioavailability(FIG. 8). Compared to scDb exhibiting a terminal half-life of 1.3 h, thescDb-IgBD fusion proteins showed a strongly prolonged circulation in theblood (FIG. 7 a). A terminal half-life of 23.3 h was determined forscDb-SpG_(C3) compared to terminal half-lives of 2.4 h forscDb-PpL_(C4*), 4.2 h for scDb-SpA_(EZ4), 9 h for scDb-SpA_(D), and 11.8h for scDb-SpA_(B) (FIG. 8). Also, scFv-IgBD fusion proteins showed astrongly prolonged circulation in the blood (FIG. 7 b). A terminalhalf-life of 20.8 h was determined for scFv-SpG_(C3) compared toterminal half-lives of 1 to 5 h for scDb-SpA_(B), scDb-SpA,scDb-SpA_(EZ4), and scDb-Pp the active compound L_(C4*).

Example 8 IL-2 Release Assay

The scDb-fusion proteins were analyzed in vitro for their ability toinduce IL-2 release (FIG. 9). Peripheral blood mononuclear cells (PBMC)from healthy donors were isolated from buffy coat as described before(Müller et al. (2007) J. Biol. Chem. 282:12650-12660). 1×10⁵ LS174Tcells/100 μl/well were seeded in 96-well plates. The next daysupernatant was removed and 150 μl of recombinant antibody added. After1 h preincubation at 37° C., 2×10⁵ PBMC/50 μl/well were added. PBMCs hadbeen thawed the day before and seeded on a culture dish. Only cells thatremained in suspension were used for the assay. After addition of PBMCs,the 96-well plate was incubated for 24 h at 37° C., 5% CO₂. Plates werecentrifuged and cell-free supernatant collected. Concentration of humanIL-2 in the supernatant was determined using the DuoSet IL-2 ELISA kit(R&D Systems) following the manufacturer's protocol. Compared with theunmodified scDb, the scDb-SpA_(B), scDb-SpA_(D) and scDb-SpA_(EZ4)fusion proteins showed a strongly increased IL-2 release in the absenceof human IgG which indicates that the SpA_(B) domain induces activationof PBMCs (FIG. 9). In contrast, scDb-SpG_(C3) did not induce an IL-2release exceeding the IL-2 release induced by scDb.

Example 9 SpG_(C3) Mutants

Variants of scDb-SpG_(C3) lacking the binding site to the Ig Fc fragment(scDb-SpG_(C3-Fab)) or the Ig Fab fragment (scDb-SpG_(C3-Fc)) wereproduced in stably transfected HEK293 cells, purified by IMAC andanalyzed for binding to human IgG, IgG-Fab fragments and IgG-Fcfragments (FIG. 10 a). Binding to the human Fc-fragment could beobserved for scDb-SpG_(C3) and scDb-SpG_(C3-Fc) whilst scDb-SpG_(C3-Fab)was not able to bind to huIgFc. In contrast, binding to the humanFab-fragment could be observed for scDb-SpG_(C3) and scDb-SpG_(C3-Fab)whilst scDb-SpG_(C3-Fc) was not able to bind to huIgFab (FIG. 10 b).scDb-SpG_(C3-Fab) was further analyzed for plasma half-life in CD1 miceas described in Example 7. A terminal half-life of 21.2±5.6 h (n=3) wasdetermined for scDb-SpG_(C3-Fab) (FIG. 10 c and d), demonstrating thatbinding to the Fab fragment of immunoglobulins is sufficient to retainthe long half-life of the wild-type fusion protein (terminal half-life23.3±5.9 h (n=6)).

Example 10 A SpG_(C3)-Diabody-scTRAIL Fusion Protein

A SpG_(C3)-diabody-scTRAIL fusion protein was generated by fusing theSpG_(C3) to an anti-EGFR diabody (VH-VL domains of a humanized anti-EGFRantibody huC225 connected by a 5 residue GGGGS linker) fused by anadditional linker to a single-chain derivative of human TRAIL (FIGS. 11and 13). For purification and detection, the protein contains a FLAG-tagat the N-terminus. The fusion protein was produced by stably transfectedHEK293 cells and purified by anti-FLAG affinity chromatography from cellculture supernatant. Westernblot with an anti-TRAIL antibody or ananti-FLAG antibody showed a single band of approximately 100 kDa,corresponding to the expected molecular mass. The purified fusionprotein showed strong binding to human IgG in ELISA demonstrating thatthe SpG_(C3) domain is also functional when fused to the N-terminus of aprotein.

Example 11 Comparison of the Pharmacokinetic Properties of scDb-SpG_(C3)and scDb-ABD_(H)

Similar to IgBDs, an albumin-binding domain (ABD) derived fromStreptococcus protein G has been shown to strongly improve the half-lifeof small recombinant proteins by recycling via the FcRn when bound toalbumin (Stork et al., 2007, Protein Eng. Des. Sel., 20, 569-576;Andersen et al., 2010, J. Biol. Chem. 286, 5234-5241). Several mutantsof the ABD with altered affinity for mouse and human albumin have beendescribed and tested for their half-life extension properties (Jonssonet al., 2008, Protein Eng. Des. Sel. 21, 515-527; Hopp et al., 2010,Protein Eng. Des. Sel. 23, 827-834). Amongst them, ABD_(H)(albumin-binding domain with high affinity) has proven to show the bestpharmacokinetic properties and seems therefore a suitable fusion proteinfor comparison. Furthermore, the affinity of the ABD_(H) towards albuminis similar to the affinity of SpG_(C3) towards IgG (Hopp et al., 2010,Protein Eng. Des. Sel. 23, 827-834). Therefore, we compared the plasmahalf-lives of scDb-ABD_(H) and scDb-SpG_(C3) after a single dose i.v.injection (25 μg/animal) into CD1 mice (FIG. 16 a and b), as describedabove in Example 7. Over the first 24 hours, scDb-SpG_(C3) showed asignificantly (p<0.01) increased plasma concentration, displayed by anAUC of 997±79% h compared to 836±81% h for the scDb-ABD_(H) and 56±15% hfor unmodified scDb (FIG. 16 c). Additionally, initial plasma half-lifewas calculated (using the first 3 values) and revealed a 1.6-foldincrease in t_(1/2)α for scDb-SpG_(C3) (2.4±0.7 h) compared toscDb-ABD_(H) (1.5±0.5 h), which was significantly different (p<0.05)(FIG. 16 d). Further investigating the biphasic profile of the fusionprotein pharmacokinetics resulted in a very similar terminal plasmahalf-life of 20.6±11.5 h for scDb-ABD_(H) and 21.0±4.8 h forscDb-SpG_(C3), calculated from 6 h to 24 h. This finding indicates thatSpG_(C3) fusion proteins compared to ABD_(H) fusion proteins, have animproved initial distribution phase resulting in an increasedbioavailability as measured by the AUC.

Example 12 Comparison of IL-2 Release by scDb-SpG_(C3-Fab) andscDb-ABD_(H) in the Absence or Presence of Albumin or IgG

The potential of the bispecific anti-CEA×anti-CD3 scDb fusion proteinsto stimulate T cells was analyzed using an IL-2 release assay. Followingthe protocol described above in Example 8, scDb-SpG_(C3-Fab) andscDb-ABD_(H) were used in different protein concentrations ranging from0.1 nM to 31.6 nM (FIG. 17). While the unmodified scDb as referencemolecule showed no or only a marginal reduction in IL-2 release in thepresence of IgG or human serum albumin (using 1/50 of the physiologicalconcentrations), scDb-ABD_(H) showed a strong reduction in signal whenpreincubated with HSA. In contrast, the SpG_(C3) variant SpG_(C3-Fab),lacking the binding site for the Ig Fc fragment, showed strongactivation even in the presence of IgG similar to the unmodified scDb,demonstrating that SpG_(C3-Fab) is especially suitable for half-lifeextension of bispecific molecules retargeting effector T cells and thatthis domain is superior over the established ABD domain.

Sequence Listing—Free Text Information

SEQ ID NO: 1 Amino acid sequence of the C3 domain of SpG:TTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVW TYDDATKTFTVTE SEQ ID NO: 2Amino acid sequence of C3-Fc:TTYKLVINGKALAGATTTKAVDAETAEKAFKQYANDNGVDGVW TYDDATKTFTVTE SEQ ID NO: 3Amino acid sequence of C3-Fab:TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVW TYDDATKTFTVTE SEQ ID NO: 4Amino acid positions 122-127 and 207-214 of a humanIg γ1 molecule according to EU index as in Kabat:Gly Pro Ser Val Phe Pro . . . Ser Asn Thr Lys Val Asp Lys LysSEQ ID NO: 5 Amino acid positions 122-127 and 207-214 of a humanIg γ2 molecule according to EU index as in Kabat:Gly Pro Ser Val Phe Pro . . . Ser Asn Thr Lys Val Asp Lys ThrSEQ ID NO: 6 Amino acid positions 122-127 and 207-214 of a humanIg γ3 molecule according to EU index as in Kabat:Gly Pro Ser Val Phe Pro . . . Ser Asn Thr Lys Val Asp Lys ArgSEQ ID NO: 7 Amino acid positions 122-127 and 207-214 of a humanIg γ4 molecule according to EU index as in Kabat:Gly Pro Ser Val Phe Pro . . . Ser Asn Thr Lys Val Asp Lys ArgSEQ ID NO: 8 Amino acid positions 122-127 and 207-214 of a mouseIg γ1 molecule according to EU index as in Kabat:Pro Pro Ser Val Tyr Pro . . . Ser Ser Thr Lys Val Asp Lys LysSEQ ID NO: 9 Amino acid positions 122-127 and 207-214 of a mouseIg γ2a molecule according to EU index as in Kabat:Ala Pro Ser Val Tyr Pro . . . Ser Ser Thr Lys Val Asp Lys LysSEQ ID NO: 10 Amino acid positions 122-127 and 207-214 of a mouseIg γ2b molecule according to EU index as in Kabat:Ala Pro Ser Val Tyr Pro . . . Ser Ser Thr Thr Val Asp Lys LysSEQ ID NO: 11 Amino acid positions 122-127 and 207-214 of a mouseIg γ3 molecule according to EU index as in Kabat:Ala Pro Ser Val Tyr Pro . . . Ser Lys Thr Glu Leu Ile Lys ArgSEQ ID NO: 12 Amino acid positions 122-127 and 207-214 of a rat Igg1 molecule according to EU index as in Kabat:Ala Pro Ser Val Tyr Pro . . . Ser Ser Thr Lys Val Asp Lys LysSEQ ID NO: 13 Amino acid sequence of scFv-SpG-C3 (anti-CEA)SEQ ID NO: 14 Amino acid sequence of scDb-SpG-C3 (anti-CEA x anti-CD3)SEQ ID NO: 15 Amino acid sequence of SpG-C3-Db-scTRAIL (anti-human EGFR)SEQ ID NO: 16 Amino acid sequence of the C1 domain of SpG: SEQ ID NO: 17Amino acid sequence of the C2 domain of SpG:

1. A complex comprising (i) an immunoglobulin (Ig) binding moiety and(ii) a pharmaceutically active moiety, wherein the Ig binding moietyspecifically binds to the constant domain 1 of the heavy chain (CH1) ofan Ig molecule.
 2. The complex of claim 1, wherein the Ig binding moietyspecifically binds to the surface-exposed region of the C_(H)1 domain ofan Ig molecule.
 3. The complex of claim 1, wherein the Ig binding moietyspecifically binds to the Fc portion of the Ig molecule.
 4. The complexof claim 1, wherein the Ig molecule is an IgG molecule, preferably anIgG1, IgG2, or IgG4 molecule.
 5. The complex of claim 1, wherein the Igbinding moiety specifically binds to an epitope formed by amino acidpositions 122-127 and/or 207-214 of an Ig molecule according to EUindex.
 6. The complex of claim 1, wherein the Ig binding moietycomprises an immunoglobulin binding domain (IgBD), preferably astreptococcus-derived IgBD.
 7. The complex of claim 1, wherein the Igbinding moiety comprises a C_(H)1 binding-IgBD of streptococcal proteinG.
 8. The complex of claim 1, wherein the Ig binding moiety comprises anamino acid sequence according to SEQ ID NO: 1 or variants thereof. 9.The complex of claim 1, wherein the pharmaceutically active moietycomprises a biological and/or chemical pharmaceutical.
 10. The complexof claim 9, wherein the biological is a pharmaceutically activepolypeptide, preferably an antigen-binding molecule.
 11. The complex ofclaim 10, wherein the antigen-binding molecule is selected from thegroup consisting of an antibody fragment, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, a heavy chain antibody, a single-domainantibody (sdAb), a single-chain variable fragment (scFv), a di-scFv, abispecific T-cell engager (BITEs), a diabody, a single-chain diabody, aDART, a triple body, an alternative scaffold protein, and a fusionprotein thereof.
 12. The complex of claim 10, wherein saidantigen-binding molecule further comprises a radioactive moiety, acytotoxic drug, a chelating moiety, a photosensitizer, or an imagingreagent.
 13. The complex of claim 11, wherein said antigen bindingmolecule is a fusion protein, which further comprises a proapoptoticprotein, an immuno-(co)stimulatory protein, immuno-suppressive protein,a cytokine, a chemokine, a toxin, a growth factor or an enzyme,preferably an RNase, a prodrug-converting enzmye, or a kinase.
 14. Thecomplex of claim 1, wherein the Ig binding moiety and thepharmaceutically active moiety are connected via covalent ornon-covalent bond(s).
 15. The complex of claim 1, wherein the Ig bindingmoiety and the pharmaceutically active moiety are connected directly orindirectly via one or more linkers.
 16. The complex of claim 14, whereinthe one or more linkers comprise peptide linkers, preferably flexiblepeptide linkers.
 17. The complex of claim 16, wherein the one or morepeptide linker comprise one or more cleavage sites, preferably one ormore endopeptidase cleavage sites.
 18. A nucleic acid moleculecomprising a sequence encoding the complex of claim
 1. 19. A vectorcomprising the nucleic acid of claim
 18. 20. A cell comprising thecomplex of claim 1, the nucleic acid of claim 18 and/or the vector ofclaim
 19. 21. A pharmaceutical composition comprising the complex ofclaim 1, the nucleic acid of claim 18 and/or the vector of claim
 19. 22.The pharmaceutical composition of claim 21, which further comprises apharmaceutically acceptable carrier and/or excipient and optionally oneor more additional active substances.
 23. A method of extending theserum half-life of an agent comprising combining that agent with thecomplex of claim
 1. 24. A method of treating a subject by administrationof a medicament comprising the complex of claim 1 to a subject in needthereof.